Detection of nucleic acid differences using combined endonuclease cleavage and ligation reactions

ABSTRACT

The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.

This application is a divisional of U.S. patent application Ser. No.09/998,481, filed Nov. 30, 2001, which claims benefit of U.S.Provisional Patent Application Ser. No. 60/250,435, filed Dec. 1, 2000,which are hereby incorporated by reference in their entirety.

The present invention arose from research sponsored by the NationalInstitutes of Health under Grant Nos. ROI-CA 65930 and ROI-CA 81467. TheUnited States Government may have certain rights.

FIELD OF THE INVENTION

The present invention relates to detecting nucleic acid differencesusing combined endonuclease (“endo”) cleavage and ligation reactions.

BACKGROUND OF THE INVENTION

Detection of DNA Sequence Variation

There is a great need in both basic and clinical research to identifyDNA sequence variations with high efficiency and accuracy. The currenttechniques for detection of such variation can be divided into twogroups: 1) detection of known mutations or polymorphisms and 2)detection of unknown mutations or polymorphisms (also referred to asmutation scanning). A variety of effective methods have been developedfor detecting known mutations and polymorphisms and include techniquessuch as direct DNA sequencing, allele-specific oligonucleotidehybridization, allele-specific PCR, DNA arrays, and PCR/LDR. There are avariety of techniques for detecting unknown mutations, but theirsensitivity and accuracy vary greatly.

Comparison of High-Throughput Techniques to Identify Unknown Mutationsin Clinical Samples.

Identifying unknown mutations in clinical samples presents similardifficulties as screening for known mutations, as well as some novelcomplications. A mutation present in a tumor sample may represent aslittle as 15% of the DNA sequence for that gene due to stromalcontamination. Therefore, screens for unknown mutations require highsensitivity in order to identify low abundance sequence. Since mostcancer genes contain multiple exons which may be altered, even forcommonly mutated genes (e.g. PTEN), most assay results of a single exonwill be negative. However, by pooling samples together, the probabilityof finding a significant mutation in a given assay increases. In orderto increase the capacity of a screen by pooling samples, the techniquemust have a high enough sensitivity to tolerate further mutationdilution which results from pooling. Further, for uncommon germlinemutations, the ability to pool samples greatly improves the throughputof evaluating large numbers of samples in multiple exons.

Other significant complications associated with screening for unknowncancer mutations are the need to: (i) Identify either frameshift,nonsense, or missense mutations, and (ii) distinguish missense mutationsfrom germline (i.e. silent) polymorphisms. The latter is of greatsignificance, because it is estimated that polymorphisms existapproximately once every kb in the human genome. Wang, D. G., et al.,Science, 280(5366):1077-82 (1998), Li, W. H., et al., Genetics,129(2):513-23 (1991), Lai, E., et al., Genomics, 54(1):31-38 (1998),Nickerson, D. A., et al., Nat Genet, 19(3):233-40 (1998), Harding, R.M., et al., Am. J. Hum. Genet., 60(4):772-89 (1997), Taillon-Miller, P.et al., Genome Res., 8(7): 748-54 (1998), Halushka, M. K., et al., Nat.Genet., 22(3): 239-47 (1999), and Cargill, M., et al., Nat. Genet.,22(3):231-38 (1999). Separating out the apparently less interestingpolymorphisms should significantly increase the efficiency inidentifying informative mutations. Unfortunately, present methods ofidentifying additional low frequency mutations with clinicalsignificance are restricted in their applications. Most methods to datelack either the accuracy to discriminate or the sensitivity to be anefficient technique. As a result, there is an urgent need for a scanningmethod with the potential to identify precise mutations and with thesensitivity to analyze tumors or germline DNA in pooled samples.

Direct Sequencing and Variation Detection Arrays.

A variety of methods have been developed to scan for unknown mutations.Direct sequencing represents an ideal in that it can detect any mutationand its position, although high throughput is costly. Gyllensten, U. B.et al., Proc. Natl. Acad. Sci. USA, 85(20): 7652-56 (1988), Hultman, T.,et al., Nucleic Acids Res., 17(13):4937-46 (1989), Phear, G. A. et al.,Methods Mol. Biol., 31:247-56 (1994), Rao, V. B., Anal Biochem.,216(1):1-14 (1994), Kovach, J. S., et al., J. Natl. Cancer Inst.,83(14):1004-9 (1991). This method has low sensitivity which prevents anaccurate analysis of pooled samples, and, in regard to solid tumorsamples, the technique is vulnerable to stromal contamination. Inblinded studies, direct sequencing was unable to identify 20% of K-rasmutations in microdissected tumor samples. This number is consistentwith other studies in which 24% of p53 mutations in lung tumor sampleswere not identified. Ahrendt, S., et al., Proc. Natl. Acad. Sci. USA,96:7382-87 (1999). When automated sequence analysis was performed onthese same lung tumor samples, this false negative value rose to 32%.Ahrendt, S., et al., Proc. Natl. Acad. Sci. USA, 96:7382-87 (1999).These results reflect the limits of sensitivity associated with directsequencing and further demonstrate the difficulty of automating thisapproach. If a sample is known to have a mutation in a specific area,then repeated attempts of direct sequencing should be able to identifythe mutation and its position. Therefore, direct sequencing may haveutility as a second step to identify the exact base changed in a generegion previously identified as containing a mutation.

Variation detection arrays (VDA) use standard hybridization microarraysto scan large sequence blocks in given genes. Despite the high scanningcapacity levels, this approach has some characteristics that limits itsutility. VDA is unable to detect all mutations and has particulardifficulty in detecting frameshift mutations, for example in the BRCA2and p53 genes. Ahrendt, S., et al., Proc. Natl. Acad. Sci. USA,96:7382-87 (1996), Hacia, J. G., et al., Nature Genetics, 14(4): 441-47(1999). Primer extension arrays also fail to detect slippage ofmononucleotide repeat sequences. Syvanen, A. C. et al., Hum. Mutat.,3(3):172-79 (1994). High false positive rates of 11-21% have beenobserved using VDA. Halushka, M. K., et al., Nat. Genet., 22(3):239-47(1999). One consequence of direct hybridization that may account forthese inaccurate results involves the disruption of secondary structure.A perfect match PCR fragment may assume a secondary structure that isnot present in variant fragments. Since a secondary structure is usuallyenergetically unfavorable with respect to fragment/array hybridizations,the variant fragment may bind the perfect match complement on the arraywith higher binding affinity than the true perfect match fragment.Hacia, J., Nature Genetics (Supplement), 21:42-47 (1999). Suchillegitimate hybridizations would produce a false positive signal.Direct hybridization of mutation-containing PCR fragments to sequenceson the array has the additional difficulty of simultaneously assayingsequence tracts with localized regions of both high G+C and A+T content.Hacia, J., Nature Genetics (Supplement), 21:42-47 (1999). Certainmutations within these tracts can significantly decrease the Tm and thuslead to false negative signals.

Gel-Based Assays, Mismatch Cleavage Enzymes, and Protein TruncationAssays

Other methods that are widely used to detect unknown mutations resolvehomoduplex and heteroduplex DNA based on their differing electrophoreticmigration behavior. These methods include single-stranded conformationalpolymorphism (SSCP) (Suzuki, Y., et al., Oncogene, 5(7):1037-43 (1990),Makino, R., et al., PCR Methods Appl., 2(1):10-13 (1992), Hayashi, K.,PCR Methods Appl., 1(1):34-38 (1991), Korn, S. et al., J. Clin. Pathol.,46(7):621-23 (1993)), denaturing-gradient gel electrophoresis (DGGE)(Fahy, E., et al., Nucleic Acids Research, 25(15):3102-9 (1997), Fodde,R. et al., Hum. Mutat., 3(2):83-94 (1994), Guldberg, P. et al., NucleicAcids Res., 22(5):880-81 (1994), Ridanpaa, M. et al., Hum. Mol. Genet.,2(6):639-44 (1993), Ridanpaa, M., et al., Mutat. Res., 334(3): 357-64(1995)), constant denaturing capillary electrophoresis (CDCE) (Chen, J.et al., Environ. Health Perspect, 3:227-29 (1994), Khrapko, K., et al.,Nucleic Acids Res., 22(3):364-69 (1994)), dideoxy fingerprinting (ddF)(Sarkar, G., et al., Genomics, 13:441-43 (1992)), and restrictionendonuclease fingerprinting (REF) (Liu, Q. et al., Biotechniques,18(3):470-77 (1995)). A similar approach, denaturing high-performanceliquid chromatography (DHPLC), also resolves homoduplex fromheteroduplex DNA but is based on separation by ion-pair reverse-phaseliquid chromatography on alkylated nonporous (styrene divinylbenzene)particles. Underhill, P. A., et al., Genome Res., 7(10):996-1005 (1997).Although these techniques contain some very desirable characteristics,none of them are complete with respect to both previously-discussedthroughput and sensitivity. The techniques which can identify theposition of the polymorphism (ddF and REF), are not applicable forevaluating low level mutations in pooled samples. The rest of thesetechniques tend to be rapid and can detect low level mutations, but theycannot distinguish missense from silent polymorphisms. In addition,since these methods do not locate the position of the mutation, they areless compatible with follow up techniques such as direct sequencing.

A sophisticated approach for detecting frame-shifts or terminationcodons in the APC gene uses a coupled transcription translation assayreferred to as the protein truncation test. Powell, S. M., et al.,Nature, 359(6392):235-37 (1992), Powell, S. M., et al., N. Engl. J.Med., 329:1982-87 (1993), Redston, M. S., et al., Gastroenterology,108(2):383-92 (1995), Petersen, G. M., et al., Hum. Genet., 91(4):307-11(1993), Su, L. K., et al., Cancer Res., 53(12):2728-31 (1993). This iscurrently the most robust approach for finding mutations which generatetruncated proteins in large genes; however, it does not detect missensemutations or polymorphisms. Polymorphisms may also be identified bycleavage of mismatches in DNA hybrids, such as DNA-RNA heteroduplexesvia RNase A mismatch cleavage (Winter, E., Proc. Natl. Acad. Sci. USA,82(22):7575-79 (1985), Perucho, M., et al., Cancer Cells, 7:137-41(1989), Myers, R. M., et al., Science, 230(4731):1242-46 (1985)), aswell as in DNA-DNA homoduplexes via chemical mismatch cleavage (CCM)(Cotton, R. G. H., et al., Proc. Natl. Acad. Sci. USA, 85:4397-401(1988), Hansen, L. L., et al., “Sensitive and Fast Mutation Detection bySolid-Phase Chemical Cleavage”, in PCR Primer: A Laboratory Manual, C.W. Diefenbach and G. S. Dveksler, Editors., Cold Spring HarborLaboratory Press: New York. p. 275-86 (1995), Haris, I. I., et al., PCRMethods Appl., 3(5):268-71 (1994)), T4 Endonuclease VII or MutY cleavage(Youil, R., et al., Proc. Natl. Acad. Sci. USA, 92(1):87-91 (1995), Xu,J. F., et al., Carcinogenesis, 17(2):321-26 (1996), Giunta, C., et al.,Diagn. Mol. Pathol., 5(4):265-70 (1996)), or via Cleavase. Recently, aplant endonuclease, CEL I, with similar activity to T4 endonuclease VIIhas been described. Oleykowski, C. A., et al., Nucleic Acids Res.,26(20):4597-602 (1998). CEL I has similar activity to nuclease S1 andworks at neutral pH. Its cleavage efficiency and background variesaccording to the mismatch and specific template examined, and furtherevaluation of other templates (e.g. in GC-rich regions) is stillrequired. The most accepted mismatch cleavage approaches identify theapproximate position of the polymorphism by using T4 Endonuclease VII orMutY to cleave a heteroduplex of normal and polymorphic substrate at amismatch. Youil, R., et al., Proc. Natl. Acad. Sci. USA, 92(1):87-91(1995), Xu, J. F., et al., Carcinogenesis, 17(2):321-26 (1996), Giunta,C., et al., Diagn. Mol. Pathol., 5(4):265-70 (1996). These enzymaticcleavage approaches identify the approximate position of mostpolymorphisms; however, these enzymes often nick matched DNA causing ahigh background noise. This high background tends to limit theirusefulness with respect to solid tumor studies.

The present invention is directed to overcoming the above deficienciesin the art.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a method foridentifying a mutant nucleic acid sequence differing by one or moresingle-base changes, insertions, or deletions, from a normal targetnucleotide sequences. This method involves blending: (1) a samplepotentially containing the normal target nucleotide sequence as well asthe mutant nucleic acid sequence; (2) providing two labeledoligonucleotide primers suitable for hybridization on complementarystrands of the target nucleotide sequence and the mutant nucleic acidsequence; and (3) providing a polymerase to form a polymerase chainreaction mixture. The polymerase chain reaction mixture is subjected toone or more polymerase chain reaction cycles comprising a hybridizationtreatment where oligonucleotide primers can hybridize to the targetnucleotide sequence and/or the mutant nucleic acid sequence, anextension treatment where the hybridized oligonucleotide primer isextended to form an extension product complementary to the targetnucleotide sequence and/or the mutant nucleic acid sequence to which theoligonucleotide primer is hybridized, and a denaturation treatment wherehybridized nucleic acid sequences are separated. After the polymerase isinactivated, the polymerase chain reaction extension products aredenatured and the polymerase chain reaction extension products areannealed to form heteroduplexed products potentially containing thenormal target nucleotide sequence and the mutant nucleic acid sequence.An endonuclease, which preferentially nicks or cleaves heteroduplexedDNA at a location one base away from mismatched base pairs, is thenblended with the heteroduplexed products to form an endonucleasecleavage reaction mixture. The endonuclease cleavage reaction mixture isblended so that the endonuclease preferentially nicks or cleavesheteroduplexed products at a location one base away from mismatched basepairs. A ligase and the potentially nicked or cleaved heteroduplexedproducts are blended to form a ligase resealing reaction mixture. Theligase resealing reaction mixture is incubated to seal the nickedheteroduplexed products at perfectly matched base pairs but withsubstantially no resealing of nicked heteroduplexed products atlocations adjacent to mismatched base pairs. The products resulting fromincubating the ligase resealing reaction mixture are separated by sizeor electrophoretic mobility, and the presence of the normal targetnucleotide sequence and the mutant nucleic acid sequence targetnucleotide are detected in the sample by distinguishing the separatedproducts resulting from incubating the ligase resealing reactionmixture.

Another aspect of the present invention relates to a method foridentifying a mutant nucleic sequence differing by one or moresingle-base changes, insertions, or deletions from a normal targetnucleic acid sequence. In this method, a sample potentially containingthe mutant nucleic acid sequence but not necessarily the normal targetnucleic acid sequence, a standard containing the normal target nucleicacid sequence, two labeled oligonucleotide primers suitable forhybridization on complementary strands of the mutant nucleic acidsequence, and a polymerase are blended to form a first polymerase chainreaction mixture. The first polymerase chain reaction mixture issubjected to one or more polymerase chain reaction cycles which includesa hybridization treatment, where the labeled oligonucleotide primers canhybridize to the mutant nucleic acid sequence, an extension treatment,where the hybridized oligonucleotide primer is extended to form anextension product complementary to the mutant nucleic acid sequence towhich the oligonucleotide primer is hybridized, and a denaturationtreatment, where hybridized nucleic acid sequences are separated. Thepolymerase is then inactivated. The normal target nucleic acid sequence,the labeled oligonucleotide primers, and the polymerase are blended toform a second polymerase chain reaction mixture. The second polymerasechain reaction mixture is subjected to one or more polymerase chainreaction cycles comprising a hybridization treatment, where the labeledoligonucleotide primers can hybridize to the normal target nucleic acidsequence, an extension treatment, where the hybridized oligonucleotideprimer is extended to form an extension product complementary to thenormal target nucleic acid sequence to which the oligonucleotide primeris hybridized, and a denaturation treatment, where hybridized nucleicacid sequences are separated. The polymerase is then deactivated. Thefirst and second polymerase chain reaction extension products aredenatured and then annealed to form heteroduplexed products potentiallycontaining the normal target nucleic acid sequence and the mutantnucleic acid sequence. An endonuclease which preferentially nicks orcleaves heteroduplexed DNA at a location one base away from mismatchedbase pairs is blended with the heteroduplexed products to form anendonuclease cleavage reaction mixture. The endonuclease cleavagereaction mixture is incubated so that the endonuclease preferentiallynicks or cleaves heteroduplexed products at a location one base awayfrom mismatched base pairs. A ligase and the potentially nicked orcleaved heteroduplexed products are blended to form a ligase resealingreaction mixture which is incubated to seal the nicked heteroduplexedproducts at perfectly matched base pairs but with substantially noresealing of nicked heteroduplexed products at locations adjacent tomismatched base pairs. The products resulting from incubating the ligaseresealing reaction mixture by size or electrophoretic mobility areseparated, and the presence of the normal target nucleic acid sequenceand the mutant nucleic acid sequence target nucleotide in the sample isdetected by distinguishing the separated products resulting fromincubating the ligase resealing reaction mixture.

Another aspect of the present invention relates to a thermostableendonuclease which generates ends that are suitable for ligation whennicking perfectly matched DNA and preferentially nicks or cleavesheteroduplexed DNA as follows: (1) at a location where base pairs aremismatched or one base beyond the mismatch and (2) at A/A, G/G, T/T,A/G, A/C, G/A, G/T, T/G, T/C, C/A, or C/T mismatched base pairs at alocation where the base pairs are mismatched or one base beyond themismatch. In each of alternatives (1) and (2), ends are generated whichare suitable for ligation when nicking perfectly matched DNA. Athermostable endonuclease in accordance with the present inventionpreferentially nicks or cleaves at least one heteroduplex formed for anysingle base mutation or polymorphism, except those having gRcg, rcRc,cgYc, or gYgy sequences, where the position of the mismatch isunderlined and shown in upper case, and generates ends which aresuitable for ligation when nicking perfectly matched DNA. Alternatively,the thermostable endonuclease, which preferentially nicks or cleavesheteroduplexed DNA, contains one, two, and three base insertions ordeletions, at a location where the base pairs are mismatched or one basebeyond the unpaired bases, and generates ends which are suitable forligation when nicking DNA at perfect matched DNA.

Another aspect of the present invention is a mutant endonuclease V(“endo V”) from Thermotoga maritima containing either: (1) a Y80Aresidue change; (2) a Y80F residue change; (3) either a Y80L, Y80I, Y80Vor Y80M residue change; (4) an R88A residue change; (5) an R88L, R88I,R88V, or R88M residue change; (6) an R88K residue change; (7) an R88N orR88Q residue change; (8) an R88D or R88E residue change; (9) an R88T orR88S residue change; (10) a E89A residue change; (11) a E89L, E89I,E89V, or E89M residue change; (12) a E89D residue change; (13) a E89N orE89Q residue change; (14) a E89R or E89K residue change; (15) a E89T orE89S residue change; (16) a H116A residue change; (17) a H116L, H116I,H116V, or H116M residue change; (18) a H116K or H116R residue change;(19) a H116N or H116Q residue change; (20) a H116T or H116S residuechange; (21) a K139A residue change; (22) a K139L, K139I, K139V, orK139M residue change; (23) a K139R residue change; (24) a K139N or K139Qresidue change; (25) a K139D or K139E residue change; (26) a K139T orK139S residue change; (27) a D43A residue change; (28) a D43E residuechange; (29) a D105A residue change; (30) a D105E residue change; (31)an F46A residue change; (32) an F46Y residue change; (33) an F46L, F46I,F46V, or F46M residue change; (34) an R118A residue change; (35) anR118L, R118I, R118V, or R118M residue change; (36) an R118K residuechange; (37) an R118N or R118Q residue change; (38) an R118D or R118Eresidue change; (39) an R118T or R118S residue change; (40) a F180Aresidue change; (41) a F180Y residue change; (42) a F180L, F180I, F180V,or F180M residue change; (43) a G83A residue change; (44) a G83L, G83I,G83V, or G83M residue change; (45) a G83K or G83R residue change; (46) aG83N or G83Q residue change; (47) a G83D or G83E residue change; (48) aG83T or G83S residue change; (49) an I179A residue change; (50) an I179Kor I179R residue change; (51) an I179N or I179Q residue change; (52) anI179D or I179E residue change; (53) an I179T or I179S residue change;(54) a D110A residue change; or (55) an H125A residue change.

A further aspect of the present invention is directed to a mutantendonuclease V which preferentially nicks or cleaves at least oneheteroduplexed DNA, containing mismatched bases, better than a wild-typeendonuclease V.

A further aspect of the present invention is directed to a method foridentifying a mutant nucleic acid sequence differing by one or moresingle-base changes, insertions, or deletions, from a normal targetnucleic acid sequence. In this method, a sample potentially containingthe normal target nucleic acid sequence as well as the mutant nucleicacid sequence, two labeled oligonucleotide primers suitable forhybridization on complementary strands of the target nucleic acidsequence and the mutant nucleic acid sequence, and a polymerase areblended to form a polymerase chain reaction mixture. The polymerasechain reaction mixture is subjected to one or more polymerase chainreaction cycles comprising a hybridization treatment, whereoligonucleotide primers can hybridize to the target nucleic acidsequence and/or the mutant nucleic acid sequence, an extensiontreatment, where the hybridized oligonucleotide primer is extended toform an extension product complementary to the target nucleic acidsequence and/or the mutant nucleic acid sequence to which theoligonucleotide primer is hybridized, and a denaturation treatment,where hybridized nucleic acid sequences are separated. After thepolymerase is inactivated, the polymerase chain reaction extensionproducts are denatured and annealed to form heteroduplexed productspotentially containing the normal target nucleic acid sequence and themutant nucleic acid sequence. An endonuclease which preferentially nicksor cleaves heteroduplexed DNA at a location one base away frommismatched base pairs and the heteroduplexed products are blended toform an endonuclease cleavage reaction mixture which is incubated sothat the endonuclease preferentially nicks or cleaves heteroduplexedproducts at a location one base away from mismatched base pairs. Aligase and the potentially nicked or cleaved heteroduplexed products areblended to form a ligase resealing reaction mixture which is incubatedto seal the nicked heteroduplexed products at perfectly matched basepairs but with substantially no resealing of nicked heteroduplexedproducts at locations adjacent to mismatched base pairs. A polymerasewith 3′-5′ exonuclease activity and the potentially nicked or cleavedheteroduplexed products are blended to form a polymerase exonucleolyticdegradation reaction mixture which is incubated under conditionseffective for the 3′-5′ exonucleolytic activity to remove several bases3′ to the nick. After the polymerase with 3′-5′ exonuclease activity isinactivated, a polymerase without 3′-5′ activity and the incubatedpolymerase degradation reaction mixture, labeled dideoxyterminatortriphosphate nucleotides, and deoxyribonucleotide triphosphates areblended to form a polymerase mini-sequencing reaction mixture which isincubated under conditions effective for the polymerase without 3′-5′activity to extend the 3′ end of the nicked or cleaved heteroduplexedproducts to form mini-sequencing reaction products. The mini-sequencingproducts are separated by size or electrophoretic mobility, and thepresence of normal target nucleic acid sequence and the mutant nucleicacid sequence are detected by distinguishing the separatedmini-sequencing products resulting from incubating the polymerasemini-sequencing reaction mixture.

The present invention has proven effective in identifying unknownframeshift, nonsense, and missense mutations as demonstrated in testsscreening for various mutations in six genes associated with thedevelopment of inherited or sporadic cancers. Furthermore, pairedgermline DNA can be evaluated side-by-side with tumor DNA, allowing thisinvention to even distinguish missense mutations from nearby or adjacentinherited polymorphisms. The assay also has broad applicability withrespect to types of sequence variations that can be detected, and canidentify 98% of the typical mutations or polymorphisms found in thehuman genome. In addition, it is capable of scanning for mutations in aregion as long as 1.7 kb or greater. The present invention can detectmutations in a high background of normal sequence. The present inventioncan detect a mutation or polymorphism in a 1:20 dilution with wild-typeDNA and can even detect mutations/polymorphisms in cases as low as 1:50dilution with wild-type DNA. This high sensitivity makes the presentinvention amenable to pooled samples and, therefore, significantlyincreasing its throughput capabilities.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram, illustrating the procedure for mutationscanning with the combination of Thermotoga maritima (“Tma”) endoV andTsp.AK16D DNA ligase.

FIGS. 2A-B show the purification of Tma endonuclease V. FIG. 2A is a12.5% SDS-PAGE, while FIG. 2B shows gel filtration of the purifiedendoV. Molecular weight markers were from Sigma Chemical (St. Louis,Mo.). Ve is the elution volume and V₀ is the void volume.

FIGS. 3A-D show cleavage assays of Tma endonuclease V on double-strandedinosine-containing substrates. FIG. 3A is an assay design where the topstrand (SEQ. ID. No. 1) is Fam labeled and the bottom strand (SEQ. ID.No. 2) is Tet labeled to allow fluorescence detection on a GeneScan gel(Perkin Elmer). The positions of nucleotide changes at both strands areunderlined. The length of the substrates and predominant products aremarked. The cleavage sites are marked by arrows. The cleavage reactionswere performed as described in M&M in the presence of 5 mM MgCl₂ withdifferent E:S (enzyme:substrate) ratios. In FIG. 3B, E:S=1:10, and therelationship between substrate and product is shown by the arrow. InFIG. 3C, E:S=10:1 and the first lanes of FIGS. 3B and C are substratenegative controls. FIG. 3D shows the location of cleavage sites. Thelength markers were synthetic oligonucleotides identical to the topstrand or the bottom strand as shown in FIG. 3A.

FIGS. 4A-B are plots of relative fluorescence intensity of cleavageproducts v. concentration of MgCl₂ which demonstrate the base-mismatchcleavage activity of Tma endonuclease V at varying concentrations ofMg²⁺ or Mn²⁺.

FIGS. 5A-D demonstrate the ability of Tma endonuclease V to cleave avariety of different single base mismatches in the presence of Mg²⁺ orMn²⁺.

FIGS. 6A-B show the cleavage and binding of AP and uracil sites.

FIG. 6A shows the cleavage of double-stranded oligonucleotidescontaining a basic site (AP site) or deoxyuridine. 1: untreatedsubstrate control; 2: AP substrates incubated at 95° C. for 1 min in 50mM NaOH as an AP site control; 3 and 10: oligonucleotide length markers;4 and 7: 1 nM Tma endoV (E:S=:10); 5 and 8: 10 nM Tma endoV (E:S=1:1); 6and 9: 100 nM Tma endoV (E:S=10:1. FIG. 6B is a gel mobility shift assayof AP and uracil sites. Reactions were performed with 20 nMdouble-stranded oligonucleotide DNA substrates, 5 mM CaCl₂ and indicatedamount of Tma endoV as specified below. Other buffer components areunchanged, 1: substrate control; 2: 10 nM Tma endoV; 4 and 6: 100 nM TmaendoV; 7: inosine substrate control; 8: 100 nM Tma endoV with 20 nMinosine substrate as positive control. FIGS. 6C-G show single strandedand nonspecific cleavage and binding by Tma endonuclease V. FIG. 6Cshows the cleavage of single-stranded oligonucleotides. Cleavagereactions were performed in the presence of 5 mM MgCl₂ or 1 mM MnCl₂with 10 nM Tma endoV (E:S=1:1). NS: non-specific sequence. FIG. 6D showsthe cleavage of plasmid substrate. Cleavage reactions were performedwith 10 nM pFB76 plasmid in the presence of 5 mM MgCl₂ or 1 mM MnCl₂. M:1 kb DNA ladder; lane 1: intact plasmid pFB76; lane 2: cleavage reactionperformed with 100 nM Tma endoV but without adding metal cofactor; lanes3 and 6: E:S=1:10; Lanes 4 and 7: E:S=1:1; Lanes 5 and 8: E:S=10:1.FIGS. 6E-G show the binding of single-stranded inosine substrate. Thebinding reaction mixture contained 100 nM single-stranded inosinesubstrate, 2 mM EDTA (FIG. 6E) or 5 mM MgCl₂ (FIG. 6F) or 5 mM CaCl₂(FIG. 6G), 20% glycerol, 10 mM HEPES (pH 7.4), 1 mM DTT, and 10 nM-1 μmTma EndoV. The reaction mixtures were incubated at 65° C. for 30 minbefore loading to a 6% native polyacrylamide gel. Lane 1: E:S=0, lane 2:E:S=1:10; lane 3: E:S=1.2; lane 4: E:S=1:1; lane 5: E:S=5:1; lane 6:E:S=10:1.

FIGS. 7A-B show the effect of pH on the base-mismatch cleavage activityof Tma endonuclease V.

FIG. 8 illustrates the optimal NaCl concentrations for the base-mismatchcleavage activity of Tma endonuclease V.

FIG. 9 is a plot of relative intensity of cleavage products v.concentration of DMSO which shows the effect of DMSO concentration onTma endonuclease V base-mismatch cleavage activity.

FIGS. 10A-B are plots of relative intensity of cleavage products v.concentration of betaine which show the effect of betaine concentrationon Tma endonuclease V base-mismatch cleavage activity.

FIGS. 11A-B are plots of relative intensity of cleavage products v.concentration of KCR. FIG. 11A shows that the cleavage of PCR fragmentswith Tma endo V favors low or no NaCl concentrations. FIG. 11B shows theinhibitory effects of KCl on the cleavage reaction with Tma endoV.

FIG. 12 is a plot of relative intensity v. time which relates theactivity of the Tma endoV cleavage reaction with respect to time.

FIG. 13A is a schematic diagram of the K-ras Exon 1 amplicon showingprimer labeling and predicted size of cleavage products. FIG. 13Bdemonstrates the ability of the present invention to detect pointmutations in the K-ras gene, and subsequent incubation with differentamounts of DNA ligase allows for sealing of non-specific cleavageproducts, while still retaining the correct, specific cleavage products.

FIGS. 14A-C are plots of relative intensity of fluorescence v. ratio ofmutant/wild type, demonstrating the sensitivity of the presentinvention.

FIG. 15 demonstrates the ability of the present invention to detectsmall insertion/deletion in BRCA 1 and BRCA2, and point mutations in thep53 gene.

FIGS. 16A-B collectively show that the present invention can detect amutation in a region of DNA as large as 1.7 kb. FIG. 16A is a schematicdiagram of the p53 1.7 kb amplicon showing primer labeling and predictedsize of cleavage product. FIG. 16B demonstrates the ability of thepresent invention to detect point mutations in the p53 gene, andsubsequent incubation with DNA ligase allows for sealing of non-specificcleavage products, while still retaining the correct, specific cleavageproducts.

FIGS. 17A-B show the chemical formulae for guanine and 7-deaza-guanine,respectively. FIG. 17C demonstrates the base-mismatch cleavage activityof Tma endoV when the PCR fragments contain 7-deaza-dG.

FIG. 18A is a schematic diagram of the DNA micro sequencing process.FIG. 18 B-C are sequence traces of a micro sequencing reactions.

FIG. 19 presents the results of the alignment of 13 identified andputative endo V enzymes from thermophilic and mesophilic archeabacteriaand eubacteria.

FIG. 20 shows the activities of mutant EndoV R88Q, R88E, H116Q, andH116T on synthetic substrates containing A:G and G:A mismatches. Thecleavage reactions were performed at 65° C. for 30 minute in a 20 μlreaction mixture containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol,1 mM MnCl₂, 20 nM DNA substrate, and 20 nM purified Tma endoV mutants.The reactions were terminated by adding an equal volume of GeneScan stopsolution. The reaction mixtures were then heated at 94° C. for 2 min.and cooled on ice. Three microliters of samples were loaded onto a 10%GeneScan denaturing polyacrylamide gel (Perkin Elmer, Foster City,Calif.). Electrophoresis was conducted at 1500 voltage for 1 hr using anABI 377 sequencer (Perkin Elmer). Lane 1-12 indicated the cleavageproducts as analyzed on a 10% denaturing polyacrylamide gel. As acontrol, wild-type Endo V was also assayed at 10 nM (WT, lane 9,10) and3 nM (WT¹lane 11 and 12), respectively.

FIGS. 21A-C show cleavage activities of Tma endonuclease V mutants on adouble-stranded inosine-containing substrate (I/A). Theinosine-containing strand (top strand) is Fam labeled and the oppositestrand (bottom strand) is Tet labeled. Cntl: substrate control (FIG.21A). In FIG. 21B, the cleavage reactions were performed with E:S(enzyme:substrate) ratio of 10:1 (S=10 nM) in the presence of 5 mMMgCl₂. In FIG. 21C, the cleavage reactions were performed with E:S(enzyme:substrate) ratio of 1:1 (S=10 nM) in the presence of 5 mM MnCl₂.

FIGS. 22A-C show gel mobility shift of Tma endonuclease V mutants. Cntl:substrate control. In FIG. 22A, gel mobility shift assays were performedwith an inosine-containing double-stranded substrate (I/A) using 2 mMEDTA instead of MgCl₂. In FIG. 22B, gel mobility shift assays with aninosine-containing double-stranded substrate (I/A) in the presence ofCaCl₂. For FIG. 22C, gel mobility shift assays were carried out with aninosine-containing double-stranded substrate (I/A) in the presence ofMgCl₂.

FIGS. 23A-B show gel mobility shift of Tma endonuclease V mutants withnicked inosine product. Cntl: substrate control. In FIG. 23A, gelmobility shift assays were conducted with a nicked inosine-containingdouble-stranded product (I/A) in the presence of CaCl₂. In FIG. 23B, gelmobility shift assays were performed with a nicked inosine-containingdouble-stranded product (I/A) in the presence of MgCl₂.

FIGS. 24A-B show a time-course analysis of inosine cleavage by Tma endoV mutants. The cleavage reactions were performed with E:S(enzyme:substrate) ratio of 1:10 (S=10 nM) in the presence of 5 mMMgCl₂. Reactions were terminated at specific time points for GeneScananalysis. Oval: Tma endo V.

FIGS. 25A-D show the binding and cleavage of single-stranded inosinesubstrate. Cntl: substrate control. FIG. 25A shows cleavage by Tma endoV mutants. The cleavage reactions were performed as described with E:S(enzyme:substrate) ratio of 1:1 (S=10 nM) in the presence of 5 mM MgCl₂.FIG. 25B shows the gel mobility shift of Tma endo V mutants withoutmetal cofactor. FIG. 25C shows the gel mobility shift of Tma endo Vmutants with 5 mM CaCl₂. FIG. 25D shows a gel mobility shift of Tma endoV mutants with 5 mM MgCl₂.

FIGS. 26A-D show cleavage activities of Tma endo V mutants on uracil andAP site substrates. The cleavage reactions were performed with E:S(enzyme:substrate) ratio of 10:1 (S=10 nM) in the presence of 5 mMMgCl₂. Cntl: substrate control. FIG. 26A shows the cleavage of A/Usubstrate. FIG. 26B shows the cleavage of G/U substrate. FIG. 26C showsthe cleavage of T/U substrate. FIG. 26D shows the cleavage of AP sitesubstrate (A/AP).

DETAILED DESCRIPTION OF THE INVENTION

Detecting DNA Sequence Differences

One aspect of the present invention is directed to a method foridentifying a mutant nucleic acid sequence differing by one or moresingle-base changes, insertions, or deletions, from a normal targetnucleotide sequence. This method involves blending: (1) a samplepotentially containing the normal target nucleotide sequence as well asthe mutant nucleic acid sequence; (2) providing two labeledoligonucleotide primers suitable for hybridization on complementarystrands of the target nucleotide sequence and the mutant nucleic acidsequence; and (3) providing a polymerase to form a polymerase chainreaction mixture. The polymerase chain reaction mixture is subjected toone or more polymerase chain reaction cycles comprising a hybridizationtreatment where oligonucleotide primers can hybridize to the targetnucleotide sequence and/or the mutant nucleic acid sequence, anextension treatment where the hybridized oligonucleotide primer isextended to form an extension product complementary to the targetnucleotide sequence and/or the mutant nucleic acid sequence to which theoligonucleotide primer is hybridized, and a denaturation treatment wherehybridized nucleic acid sequences are separated. After the polymerase isinactivated, the polymerase chain reaction extension products aredenatured and the polymerase chain reaction extension products areannealed to form heteroduplexed products potentially containing thenormal target nucleotide sequence and the mutant nucleic acid sequence.An endonuclease, which preferentially nicks or cleaves heteroduplexedDNA at a location one base away from mismatched base pairs, is thenblended with the heteroduplexed products to form an endonucleasecleavage reaction mixture. The endonuclease cleavage reaction mixture isblended so that the endonuclease preferentially nicks or cleavesheteroduplexed products at a location one base away from mismatched basepairs. A ligase and the potentially nicked or cleaved heteroduplexedproducts are blended to form a ligase resealing reaction mixture. Theligase resealing reaction mixture is incubated to seal the nickedheteroduplexed products at perfectly matched base pairs but withsubstantially no resealing of nicked heteroduplexed products atlocations adjacent to mismatched base pairs. The products resulting fromincubating the ligase resealing reaction mixture are separated by sizeor electrophoretic mobility, and the presence of the normal targetnucleotide sequence and the mutant nucleic acid sequence are detected inthe sample by distinguishing the separated products resulting from saidincubating the ligase resealing reaction mixture.

The first step of the invention is the preparation of heteroduplexnucleic acid fragments. In the preferred embodiment, genomic DNAcontaining both wild-type and the sequence variation(s) (e.g. singlenucleotide mutations or polymorphisms, one or more nucleotideinsertions, and one or more nucleotide deletions) is PCR amplified withlabeled oligonucleotide primers. Fluorescent, infrared, radioactive, orother labels may be used in the primers. In the preferred embodiment,Taq DNA polymerase or other PCR enzymes are inactivated, for example, bydigestion with proteinase K. The mixture of mutation or polymorphismcontaining and wild-type PCR fragments are denatured and then reannealedto form heteroduplex PCR fragments with nucleotide mismatches. In thepreferred embodiment, denaturation is achieved by heating the fragmentsabove their Tm value (generally greater than 94° C.), and reannealing isachieved by cooling first to 50-85° C., more preferably, 65° C. for 5-30minutes, more preferably 15 minutes, and then to room temperature for5-30 minutes, more preferably 15 minutes, to form heteroduplex PCRfragments. Alternative means of denaturing/renaturing may be used. Ifwild-type genomic DNA is not known to be present in the originalreaction, then concurrently in a separate reaction, wild type genomicDNA is PCR amplified using the exact same primers as above. Equal molaramounts of mutation containing PCR fragments and wild type PCR fragmentsare mixed, heated, and then cooled to form heteroduplex PCR fragmentswith nucleotide mismatches.

The second step utilizes Tma endonuclease V for cleavage of theheteroduplex DNA containing base mismatches. This reaction is preferablyperformed in an optimized reaction buffer at high temperature (50-65°C.) for 30 minutes to 1 hour. Optimal buffer conditions include aneutral pH, low or no salt, and the presence of Mg²⁺. Addition oforganic solvents or other compounds, such as DMSO and betaine, may beused to facilitate cleavage by Tma EndoV. Use of alternative conditionsor metal co-factors (such as Mn²⁺) may also facilitate cleavage. Tmaendonuclease V activity can be sufficient even under sub-optimalconditions. The cleavage site was determined to be one nucleotide beyondthe 3′ position of the nucleotide mismatch.

For the next step, a supplemental buffer is added to bring the contentsand concentration of the buffer to a level optimized for a thermostableDNA ligase. In the preferred embodiment, a Thermus species (“Tsp.”)AK16D DNA ligase is used. This ligation reaction is performed at 45 to85° C., preferably 65° C., for 2 to 60 minutes, preferably 20 minutesand utilizes the high specificity of Tsp. AKI 6D DNA ligase to resealcomplementary nicks, while leaving cleaved mismatches unaltered. Thisgreatly reduces background and, therefore, dramatically increases thesensitivity of the assay.

In the fourth step, the cleaved fragments are separated, for example, byelectrophoresis on a denaturing polyacrylamide gel or by capillaryelectrophoresis. Since the PCR primers of step one are labeled,fragments can be detected with the corresponding detection equipment. Inthe preferred embodiment, primers are fluorescently labeled and detectedusing automated DNA sequencing or fluorescent fragment analysisinstrumentation. The lengths of products are determined by comparison ofthe mobility of cleavage products to a fluorescent labeled molecularsize standard. This allows for an approximate determination of theposition of a mutation.

A second embodiment of this invention can incorporate a micro-sequencingmethod to determine which nucleotide is mutated. In preparing the PCRfragments, unlabeled primers are used in place of labeled primers, sothat the heteroduplex PCR fragments are not fluorescent. After cleavageby Tma endoV and ligation by Tsp. AK16D ligase, E. coli. DNA polymeraseI Klenow fragment is used to excise several nucleotides from the 3′ endof the nick generated by Tma endoV. Next, Thermus aquaticus (“Taq”) DNApolymerase FS extends the shortened fragment using a mixture offluorescent labeled dideoxynucleotides and unlabeled deoxynucleotides assubstrates. This results in a short sequencing ladder from which theposition and base of the variant nucleotide can be determined due to themixed signal of the normal and mutated nucleotide at the variantposition.

FIG. 1 is a schematic drawing illustrating the process of the presentinvention. In this drawing, the sample potentially contains a normaltarget nucleic acid sequence as well as a mutant nucleic acid sequencediffering by one or more single-base changes, insertions, or deletionsfrom the normal target nucleotide sequence.

In the first step of this procedure, target DNA molecules in a sample,potentially containing wild type and mutant nucleic acid sequences areamplified by a polymerase chain reaction process. This involvesdenaturation of double stranded target DNA molecules to separatecomplementary strands from one another. Primers with fluorescent labelsF1 and F2 are then caused to hybridize to part of the target DNAmolecule. In the presence of DNA polymerase and dNTPs, the primers areextended to form extension products which are complementary to a strandof the target DNA molecule. After extension is completed, the hybridizednucleic acid strands are separated from one another by a denaturationstep. This cycle of hybridization, primer extension, and denaturation isrepeated until sufficient amplification has taken place. Once this hasoccurred, PCR is terminated by inactivating the polymerase anddenaturing the hybridized nucleic acid strands.

In the second step of FIG. 1, the extension products formed by PCR areannealed to one another to form heteroduplexed products. To the extentthat these extension products were formed in the first step of FIG. 1 byextension of a primer hybridized to a mutant nucleic acid sequence inthe sample, the extension product will include a mismatched base. Asshown in the second step of FIG. 1, the mismatch in the extensionproduct can be an A, C, T, or G nucleotide. When, in the second step ofFIG. 1, an extension product produced by target nucleic acid sequenceforms a heteroduplex with an extension product produced by a mutantnucleic acid sequence, there will be a mismatch in the heteroduplex.This lack of complementation between such extension products is shown bya “bubble” where the extension products are displaced from one another.Although not shown in FIG. 1, homoduplexes containing extension productsproduced solely from mutant nucleic acid (which would usually be rare)or produced solely from the target nucleic acid sequence would be fullycomplementary, so no bubble would form. The present invention isdirected to detecting the presence of such mutant nucleic acid sequencesin the sample by analyzing for mismatches in the heteroduplexes.

The third step of FIG. 1 involves subjecting the heteroduplexed productsto an endonuclease which preferentially nicks or cleaves the componentstrands of the heteroduplexed products at a location one base away froma mismatched base. In the case of heteroduplexed products which areformed from an extension product produced by target nucleic acidsequence annealed to an extension product produced by a mutant nucleicacid sequence as shown in the third step of FIG. 1, the componentnucleic acid strands are nicked or cleaved one base away from thebubble. As also shown in the third step of FIG. 1, the endonucleasecleaves these strands at non-specific cleavage sites where there is nomismatch. As to the nicking which takes place at specific cleavage siteswhere there is a mismatch, the endonuclease is generally very effectiveat making nicks in strands where the mismatch base is an A or G, is lesseffective at making nicks where the mismatch base is T, and is generallyineffective at nicking where the mismatch base is C. This is shown inthe third step of FIG. 1 where nicking has occurred at the mismatched A,G, and T bases but not at the mismatched C base.

After endonuclease treatment is completed, the resulting potentiallynicked or cleaved heteroduplexed products are treated with a ligase. Asshown in the fourth step of FIG. 1, the ligase reseals the nickedheteroduplexed products at perfectly mismatched bases. However, there isno resealing of the nicked heteroduplexed products at locations adjacentto where there is a mismatched base.

The products of the ligase resealing step are then separated from oneanother by size or electrophoretic mobility, usually by gelelectrophoresis. The results of gel electrophoresis before treatmentwith either endonuclease or ligase, after treatment with endonucleaseand treatment with ligase, and after treatment with both endonucleaseand ligase is shown in FIG. 1. As a result, cleavage products produceddue to the presence of mutant nucleic acid sequence in the sample aredetected.

In carrying out the process of the present invention, the sample cancontain target nucleotide sequence which is either genomic DNA, DNAisolated from tumor samples, a double stranded cDNA copy of mRNA, or aPCR amplified DNA fragment. In the sample being analyzed according tothe process of the present invention, the molar ratio of the mutantnucleic acid sequence to the normal target nucleotide sequence is in arange of 1:20 to 20:1.

The process of the present invention is capable of distinguishing aninherited or sporadic mutation or polymorphism from a polymorphism inthe normal target sequence. This distinction can be made in a tumorsuppressor gene, oncogene, or DNA replication or repair gene. Such genesinclude Bcl2, Mdm2, Cdc25A, Cyclin D1, Cyclin E1, Cdk4, survivin, HSP27,HSP70, p53, p21^(Cip), p16^(Ink4a), p19^(ARF), p15^(INK4b), p27^(Kip),Bax, growth factors, EGFR, Her2-neu, ErbB-3, ErbB-4, c-Met, c-Sea, Ron,c-Ret, NGFR, TrkB, TrkC, IGF1R, CSF1R, CSF2, c-Kit, AXL, Flt-1(VEGFR-1), Flk-1 (VEGFR-2), PDGFRα, PDGFRβ, FGFR-1, FGFR-2, FGFR-3,FGFR-4, other protein tyrosine kinase receptors, β-catenin, Wnt(s), Akt,Tcf4, c-Myc, n-Myc, Wisp-1, Wisp-3, K-ras, H-ras, N-ras, c-Jun, c-Fos,PI3K, c-Src, Shc, Raf1, TGFβ, and MEK, E-Cadherin, APC, TβRII, Smad2,Smad4, Smad 7, PTEN, VHL, BRCA1, BRCA2, ATM, hMSH2, hMLH1, hPMS1, hPMS2,or hMSH3.

Since residual active Taq DNA polymerase can extend EndoV cleaved DNA,PCR reactions can be incubated with proteinase K at 45 to 75° C. for 5to 60 min., preferably 70° C. for 10 min. Subsequently, proteinase K isactivated by incubating at 90 to 95° C. for 10 to 60 minutes, preferably70° C. for 10 minutes. After amplification and proteinase K digestion,PCR fragments can be separated by agarose gel electrophoresis andvisualized via ethidium bromide staining.

Most biological sources of target DNA will contain both variant(mutation or polymorphism) and wild type DNA. In these cases, it is notnecessary to add wild-type PCR fragments exogenously to the heteroduplexhybridization step. For example, if the substrate is genomic DNAcontaining a heterozygous germline mutation, only 50% of the PCRfragments will contain a mutation, while the other half will be ofwild-type sequence. Therefore, it is not necessary to add wild-type PCRfragments. Likewise, for solid tumor samples, there is typically asignificant amount of stromal (i.e. wild-type) DNA within these samples.For sources of substrate in which a significant amount of endogenouswild-type DNA does not exist, an approximately equal amount of wild-typePCR fragments needs to be added. The optimal final ratio ofmutant-to-wild type PCR fragments should be 1:1, although the techniqueis compatible with other ratios of mutant-to-wild type PCR fragments.

The labeled oligonucleotide primers are labeled, preferably at their 5′ends. Useful labels include chromophores, fluorescent moieties, enzymes,antigens, heavy metals, magnetic probes, infrared dyes, phosphorescentgroups, radioactive materials, chemiluminescent moieties, andelectrochemical detecting moieties.

The polymerase is either a native or recombinant thermostable polymerasefrom Thermus aquaticus, Thermus thermophilus, Pyrococcus furiosus, orThermotoga maritima.

The polymerase chain reaction process is fully described in H. Erlich,et. al., “Recent Advances in the Polymerase Chain Reaction,”Science 252:1643-50 (1991); M. Innis, et. al., PCR Protocols: A Guide to Methods andApplications, Academic Press: New York (1990); and R. Saiki, et. al.,“Primer-directed Enzymatic Amplification of DNA with a Thermostable DNAPolymerase,” Science 239: 487-91 (1988), which are hereby incorporatedby reference. The polymerase chain reaction is initiated by addingeither the polymerase or metal co-factors at temperatures of 65-94° C.to the polymerase chain reaction mixture. The step of denaturing thepolymerase chain reaction extension products is carried out in thepresence of proteinase K, preferably by heating to 80 to 105° C.,preferably 94° C. The step of annealing the polymerase chain reactionextension products is carried out by cooling first to 50 to 85° C.,preferably 65° C., for 5 to 30 minutes, preferably, 10 minutes and thento room temperature for 5 to 30 minutes, preferably, 15 minutes.

For heteroduplex DNA formation, the mixture containing fluorescentlylabeled mutant and wild-type PCR fragments is denatured by heating at 93to 100° C. for 15 sec. to 5 min., preferably, 94° C. for 1 min, thusrendering the DNA single-stranded. This is followed by a re-annealingstep at 45 to 85° C. for 2 to 60 min., preferably 65° C. for 10 min,and, subsequently, incubating at room temperature for 5 to 30 min.,preferably 15 min. After this process, theoretically 50% of there-annealed products are heteroduplex DNA containing a base-mismatch. Analternative reanneal step would be a slow cool from 95 to 25° C.,decreasing the temperature by less than 1° C. per minute, preferably,from 94° C. to 65° C. for 30-60 minutes. Alternative means ofdenaturing/renaturation of the DNA (such as treatment with a basefollowed by neutralization) may also be used. Typically, the polymerasechain reaction extension products have a length in the range of 50 bp to1,700 bp.

The endonuclease is preferably an Endonuclease V from Thermotogamaritima, Aquifex aeolicus, Pyrococcus furiosus, Pyrococcus horikoshii,Pyrococcus abyssi, Pyrobaculum aerophilum, Archaeoglobus fulgidus,Aeropyrum pernix, Clostridium acetobutylicum, or Bacillus subtilis. Theendonuclease desirably nicks or cleaves heteroduplexed products at alocation on the 3′ side one base away from mismatched base pairs. Theendonuclease preferentially cleaves mismatches within the heteroduplexedproducts selected from the group consisting of A/A, G/G, T/T, A/G, A/C,G/A, G/T, T/G, T/C, C/A, and C/T. Alternatively, the endonucleasepreferentially nicks or cleaves at least one of the heteroduplexedproducts formed for any single base mutation or polymorphism, exceptthose having a sequence selected from the group consisting of gRcg,rcRc, cgYc, and gYgy, where the position of the mismatch is underlinedand shown in upper case. The endonuclease preferentially nicks orcleaves one, two, and three base insertions or deletions within theheteroduplexed products.

The endonuclease cleavage reaction is preferably carried out in presenceof MgCl₂ at a concentration of 2-7 mM or MnCl₂ at a concentration of0.4-1.2 mM. MgCl₂ should be added where the endonuclease toheteroduplexed product weight ratio in the endonuclease cleavagereaction mixture is in the range of 10:1 to 100:1; substantially no NaClor KCl is present. Where the endonuclease to heteroduplexed productweight ratio in the endonuclease cleavage reaction mixture is in therange of 1:1 to 1:10, MnCl₂ should be added; in this case, a 25 to 75mM, preferably 50 mM, concentration of NaCl or KCl is present.Endonuclease cleavage can also be carried out in the presence of DMSO ina volume percent range of 2.5% to 10% and betaine in a concentration of0.5M to 1.5M. Preferably, the endonuclease treatment is carried out at65° C. for 1 hour.

In the second step of the present invention, heteroduplexed PCRfragments are cleaved by Tma endonuclease V. Tma endonuclease V containsunique properties that make it ideal for this process. Most significantis its ability to preferentially cleave one base beyond the 3′ side of amismatch and the fact that spurious nicks at complementary regions aresuitable substrates for religation with DNA ligase. While there areother mismatch repair enzymes which are more efficient in recognizingbase mismatches, they generally do not cleave at the mismatch, nor dothey leave ends suitable for religation. In conjunction with anappropriate ligase, these properties of Tma endo V allow for thereduction of background noise due to spurious nicking, while maintainingcleaved sites associated with mismatch sequence.

The third step of this invention seals nonspecific nicks in theheteroduplex PCR fragments with a thermostable ligase, such as Thermusspecies AK16D, Thermus aquaticus, Thermus thermophilus, Pyrococcusfuriosus, or Thermotoga maritima. The thermostable ligase may be derivedfrom Thermus aquaticus. M. Takahashi, et al., “Thermophillic DNALigase,” J. Biol. Chem. 259:10041-47 (1984), which is herebyincorporated by reference. Alternatively, it can be preparedrecombinantly. Procedures for such isolation as well as the recombinantproduction of Thermus aquaticus ligase as well as Thermus themophilusligase) are disclosed in WO 90/17239 to Barany, et. al., and F. Barany,et al., “Cloning, Overexpression and Nucleotide Sequence of aThermostable DNA-Ligase Encoding Gene,” Gene 109:1-11 (1991), which arehereby incorporated by reference. These references contain completesequence information for this ligase as well as the encoding DNA. Ligaseresealing is preferably carried out in the presence of 50 mM KCl toinhibit further endonucleolytic cleavage. Preferably, Tsp AK16D ligaseis used. Ligase resealing is carried out at a pH value between 7.2 and7.8 when measured at 25° C. Ideally, the cleavage of Tma endonuclease Vshould be inhibited in this step. The optimal reaction buffer for TspAK16D ligase is 20 mM Tris-HCl (pH 8.5), 5 mM MgCl₂, 25-75 mM(preferably, 50 mM) KCl, 10 mM dithiothreitol, 1 mM NAD⁺, and 20 mg/mlBSA. See Tong, J., et al., Nucleic Acid Research 27:788-94 (1999). Asdemonstrated in the Examples, infra, Tma. endo V is almost completelyinhibited in the presence of 50 mM NaCl or 50 mM KCl.

In order to obtain near optimal buffer conditions for the Tsp AK16Dligase reaction, a supplemental buffer is added to the Tma endo Vreaction. In a preferred embodiment, the 10× supplemental bufferconsists of 200 mM Tris-HCl (pH 8.5), 12.5 mM MgCl₂, 500 mM KCl, 100 mMDTT, and 200 g/ml BSA. Typically, 15 μL of the reaction mixture from aTma endonuclease V cleavage reaction, 2 μL of 10× supplemental buffer, 1μL of 20 mM NAD⁺, and 2 μL of 10-100 nM Tsp AK16D ligase (stock enzymesolution) are combined. The mixture can then be incubated at 65° C. for20 min and terminated by adding an equal volume of GeneScan stopsolution (50 mM EDTA, 1% blue dextran and 80% formamide).

The next step involves detection of the reaction products which can becarried out using polyacrylamide gel electrophoresis or capillary gelelectrophoresis.

In the preferred embodiment, the reaction mixture is denatured at 94° C.for only 1 minute (to avoid DNA fragmentation which can increasebackground signal), and then cooled on ice. 2-3 μL of the mixture canthen be loaded onto a 6% denaturing polyacrylamide gel andelectrophoresed for 1 hour. An ABI 377 sequencer (Perkin Elmer) at 1000volt, 60 mA current, 200 W power, and a gel temperature of 45° C. can beused to separate and detect DNA products, although alternative capillaryor gel electrophoresis approaches can be used. Fluorescent groups, 6-FAM(bottom fragment) and TET (top fragment), resolve blue and green,respectively, in the ABI DNA 377 sequencer. The color of the cleavageband indicates whether the cleavage product originated from the top orbottom strand. TAMRA labeled GeneScan Molecular size standard 500 areloaded on the same gel. This allows for the molecular weight of cleavageproducts to be estimated by comparing the relative mobility of acleavage product to the size standard. Preferably, the GeneScan analysissoftware versions 2.1 or 3.0a (PE-Biosystems) is used, although anystate of the art gel-analysis software can instead be employed. Thisanalysis allows for the approximate site of the mutation to bedetermined.

Another aspect of the present invention relates to a method foridentifying a mutant nucleic sequence differing by one or moresingle-base changes, insertions, or deletions from a normal targetnucleic acid sequence. In this method, a sample potentially containingthe mutant nucleic acid sequence but not necessarily the normal targetnucleic acid sequence, a standard containing the normal target nucleicacid sequence, two labeled oligonucleotide primers suitable forhybridization on complementary strands of the mutant nucleic acidsequence, and a polymerase are blended to form a first polymerase chainreaction mixture. The first polymerase chain reaction mixture issubjected to one or more polymerase chain reaction cycles which includesa hybridization treatment, where the labeled oligonucleotide primers canhybridize to the mutant nucleic acid sequence, an extension treatment,where the hybridized oligonucleotide primer is extended to form anextension product complementary to the mutant nucleic acid sequence towhich the oligonucleotide primer is hybridized, and a denaturationtreatment, where hybridized nucleic acid sequences are separated. Thepolymerase is then inactivated. The normal target nucleic acid sequence,the labeled oligonucleotide primers, and the polymerase are blended toform a second polymerase chain reaction mixture. The second polymerasechain reaction mixture is subjected to one or more polymerase chainreaction cycles comprising a hybridization treatment, where the labeledoligonucleotide primers can hybridize to the normal target nucleic acidsequence, an extension treatment, where the hybridized oligonucleotideprimer is extended to form an extension product complementary to thenormal target nucleic acid sequence to which the oligonucleotide primeris hybridized, and a denaturation treatment, where hybridized nucleicacid sequences are separated. The polymerase is then deactivated. Thefirst and second polymerase chain reaction extension products aredenatured and then annealed to form heteroduplexed products potentiallycontaining the normal target nucleic acid sequence and the mutantnucleic acid sequence. An endonuclease which preferentially nicks orcleaves heteroduplexed DNA at a location one base away from mismatchedbase pairs is blended with the heteroduplexed products to form anendonuclease cleavage reaction mixture. The endonuclease cleavagereaction mixture is incubated so that the endonuclease preferentiallynicks or cleaves heteroduplexed products at a location one base awayfrom mismatched base pairs. A ligase and the potentially nicked orcleaved heteroduplexed products are blended to form a ligase resealingreaction mixture which is incubated to seal the nicked heteroduplexedproducts at perfectly matched base pairs but with substantially noresealing of nicked heteroduplexed products at locations adjacent tomismatched base pairs. The products resulting from incubating the ligaseresealing reaction mixture by size or electrophoretic mobility areseparated, and the presence of the normal target nucleic acid sequenceand the mutant nucleic acid sequence target nucleotide in the sample isdetected by distinguishing the separated products resulting fromincubating the ligase resealing reaction mixture.

DNA Micro-Sequencing for Identification of the Position and Compositionof a Mutation or Insertion/Deletion.

A further aspect of the present invention is directed to a method foridentifying a mutant nucleic acid sequence differing by one or moresingle-base changes, insertions, or deletions, from a normal targetnucleic acid sequence. In this method, a sample potentially containingthe normal target nucleic acid sequence as well as the mutant nucleicacid sequence, two labeled oligonucleotide primers suitable forhybridization on complementary strands of the target nucleic acidsequence and the mutant nucleic acid sequence, and a polymerase areblended to form a polymerase chain reaction mixture. The polymerasechain reaction mixture is subjected to one or more polymerase chainreaction cycles comprising a hybridization treatment, whereoligonucleotide primers can hybridize to the target nucleic acidsequence and/or the mutant nucleic acid sequence, an extensiontreatment, where the hybridized oligonucleotide primer is extended toform an extension product complementary to the target nucleic acidsequence and/or the mutant nucleic acid sequence to which theoligonucleotide primer is hybridized, and a denaturation treatment,where hybridized nucleic acid sequences are separated. After thepolymerase is inactivated, the polymerase chain reaction extensionproducts are denatured and annealed to form heteroduplexed productspotentially containing the normal target nucleic acid sequence and themutant nucleic acid sequence. An endonuclease which preferentially nicksor cleaves heteroduplexed DNA at a location one base away frommismatched base pairs and the heteroduplexed products are blended toform an endonuclease cleavage reaction mixture which is incubated sothat the endonuclease preferentially nicks or cleaves heteroduplexedproducts at a location one base away from mismatched base pairs. Aligase and the potentially nicked or cleaved heteroduplexed products areblended to form a ligase resealing reaction mixture which is incubatedto seal the nicked heteroduplexed products at perfectly matched basepairs but with substantially no resealing of nicked heteroduplexedproducts at locations adjacent to mismatched base pairs. A polymerasewith 3′-5′ exonuclease activity and the potentially nicked or cleavedheteroduplexed products are blended to form a polymerase exonucleolyticdegradation reaction mixture which is incubated under conditionseffective for the 3′-5′ exonucleolytic activity to remove several bases3′ to the nick. After the polymerase with 3′-5′ exonuclease activity isinactivated, a polymerase without 3′-5′ activity and the incubatedpolymerase degradation reaction mixture, labeled dideoxyterminatortriphosphate nucleotides, and deoxyribonucleotide triphosphates areblended to form a polymerase mini-sequencing reaction mixture which isincubated under conditions effective for the polymerase without 3′-5′activity to extend the 3′ end of the nicked or cleaved heteroduplexedproducts to form mini-sequencing reaction products. The mini-sequencingproducts are separated by size or electrophoretic mobility, and thepresence of normal target nucleic acid sequence and the mutant nucleicacid sequence are detected by distinguishing the separatedmini-sequencing products resulting from incubating the polymerasemini-sequencing reaction mixture.

Treatment with a polymerase with 3′-5′ exonuclease activity is carriedout at a temperature of 20 to 40° C. for 10 to 60 minutes, preferably37° C. and 30 minutes. Suitable polymerases with 3′-5′ exonucleaseactivity are E. coli DNA polymerase I Klenow fragment, T4 DNApolymerase, and T7 DNA polymerase.

The polymerase without 3′-5′ activity is utilized in the polymerasemini-sequencing reaction mixture at a temperature of 20 to 85° C. for 10to 60 seconds, preferably 60° C. and 30 seconds. Suitable polymeraseswithout 3′-5′ activity are AmpliTaq DNA polymerase FS (Perkin Elmer,Foster City, Calif.), Thermo-sequenase, (Amersham, Piscataway, N.J.08855), and DyNASeq, (M.J. Research, 590 Lincoln Street, Waltham, Mass.02451). Examples of a mesophilic polymerases without 3′ to 5′exonuclease activity are Sequenase (USB, Cleveland, Ohio 44128), andother polymerases with site-specific mutations to knock out the 3′ to 5′exonuclease activity.

In this embodiment of the present invention, an optional DNA sequencingstep (i.e. micro DNA sequencing) can be included to accurately determinethe composition and position of an altered nucleotide. In this variationof the present invention, the procedure is similar to that describedabove, except that unlabeled PCR primers are used in the PCR reaction.This results in PCR fragments which are unlabeled and, therefore,compatible with fluorescent dideoxysequencing. The presence or absenceof fluorescent label is not an issue with radiolabeled dideoxysequencing, and, therefore, the variation of the technique is purely oneof compatibility with the detection method of the sequencing assay andnot necessarily required for DNA sequencing in general.

The micro-sequencing strategy contains two additional steps. In thefirst added step, after the Tma endo V cleavage and ligation with Tsp.AK16D ligase, the 3′ exonuclease activity of DNA polymerase I Klenowfragment is utilized to excise a few bases 3′→5′ from the mismatch nickgenerated by Tma Endo V. E. coli DNA polymerase I Klenow fragmentpossesses approximately a hundred-fold lower 3′ exonuclease activitythan T4 or T7 DNA polymerase (Sambrook, J. et al., Molecular Cloning—ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), which is hereby incorporated by reference).This allows for a more controlled removal of only a few bases. AmpliTaqDNA polymerase FS (Perkin Elmer) is then used to fill the resulting gapwith a substrate mixture of BigDye™ ddNTP/dNTP. This results in a veryshort fluorescent sequence ladder which can be analyzed to determine theexact position and nature of the variation.

Mutants of Thermatoga maritima EndoV

Another aspect of the present invention relates to a thermostableendonuclease which generates ends that are suitable for ligation whennicking perfectly matched DNA and preferentially nicks or cleavesheteroduplexed DNA as follows: (1) at a location where base pairs aremismatched or one base beyond the mismatch and (2) at A/A, G/G, T/T,A/G, A/C, G/A, G/T, T/G, T/C, C/A, or C/T mismatched base pairs at alocation where the base pairs are mismatched or one base beyond themismatch. In each of alternatives (1) and (2), ends are generated whichare suitable for ligation when nicking perfectly matched DNA. Athermostable endonuclease in accordance with the present inventionpreferentially nicks or cleaves at least one heteroduplex formed for anysingle base mutation or polymorphism, except those having gRcg, rcRc,cgYc, or gYgy sequences, where the position of the mismatch isunderlined and shown in upper case, and generates ends which aresuitable for ligation when nicking perfectly matched DNA. Alternatively,the thermostable endonuclease, which preferentially nicks or cleavesheteroduplexed DNA, contains one, two, and three base insertions ordeletions, at a location where the base pairs are mismatched or one basebeyond the unpaired bases, and generates ends which are suitable forligation when nicking DNA at perfect matched DNA.

Another aspect of the present invention is a mutant endonuclease V fromThermotoga maritima containing either: (1) a Y80A residue change; (2) aY80F residue change; (3) either a Y80L, Y80I, Y80V or Y80M residuechange; (4) an R88A residue change; (5) an R88L, R88I, R88V, or R88Mresidue change; (6) an R88K residue change; (7) an R88N or R88Q residuechange; (8) an R88D or R88E residue change; (9) an R88T or R88S residuechange; (10) a E89A residue change; (11) a E89L, E89I, E89V, or E89Mresidue change; (12) a E89D residue change; (13) a E89N or E89Q residuechange; (14) a E89R or E89K residue change; (15) a E89T or E89S residuechange; (16) a H116A residue change; (17) a H116L, H116I, H116V, orH116M residue change; (18) a H116K or H116R residue change; (19) a H116Nor H116Q residue change; (20) a H116T or H116S residue change; (21) aK139A residue change; (22) a K139L, K139I, K139V, or K139M residuechange; (23) a K139R residue change; (24) a K139N or K139Q residuechange; (25) a K139D or K139E residue change; (26) a K139T or K139Sresidue change; (27) a D43A residue change; (28) a D43E residue change;(29) a D105A residue change; (30) a D105E residue change; (31) an F46Aresidue change; (32) an F46Y residue change; (33) an F46L, F46I, F46V,or F46M residue change; (34) an R118A residue change; (35) an R118L,R118I, R118V, or R118M residue change; (36) an R118K residue change;(37) an R118N or R118Q residue change; (38) an R118D or R118E residuechange; (39) an R118T or R118S residue change; (40) a F180A residuechange; (41) a F180Y residue change; (42) a F180L, F180I, F180V, orF180M residue change; (43) a G83A residue change; (44) a G83L, G83I,G83V, or G83M residue change; (45) a G83K or G83R residue change; (46) aG83N or G83Q residue change; (47) a G83D or G83E residue change; (48) aG83T or G83S residue change; (49) an I179A residue change; (50) an I179Kor I179R residue change; (51) an I179N or I179Q residue change; (52) anI179D or I179E residue change; (53) an I179T or I179S residue change;(54) a D110A residue change; or (55) an H125A residue change.

A further aspect of the present invention is directed to a mutantendonuclease V (and its above-described uses) which preferentially nicksor cleaves at least one heteroduplexed DNA, containing mismatched bases,better than a wild-type endonuclease V. Preferably, one of themismatched bases in at least one of the heteroduplexed DNA is “A” or“G”.

EXAMPLES Example 1 Reagents, Media, and Strains

All routine chemical reagents were purchased from Sigma Chemicals (St.Louis, Mo.) or Fisher Scientific (Fair Lawn, N.J.). deoxynucleotide,BSA, and ATP were purchased from Boehringer-Mannheim (Indianapolis,Ind.). Deoxyoligonucleotides were ordered from Integrated DNATechnologies Inc. (Coralville, Iowa). HiTrap SP columns were purchasedfrom Amersham-Pharmacia Biotech (Piscataway, N.J.).

Restriction enzymes, T4 DNA ligase and DNA polymerase I (Klenowfragment) were purchased from NewEngland Biolab (Beverly, Mass.). DNAsequencing kits, PCR kits, and GENESCAN-500 (TAMRA) Size Standard werepurchased from Applied Biosystems Division of Perkin-Elmer Corporation(Foster City, Calif.). Pfu DNA polymerase, PCR buffer and TaqPlusPrecision PCR kit were purchased from Stratagene (La Jolla, Calif.).Protein assay kit was obtained from Bio-Rad (Hercules, Calif.).

FB medium (one liter) consisted of 25 gram Bacto tryptone, 7.5 gramyeast extract, 6 gram NaCl, 1 gram glucose, and 50 ml of 1 M Tris-HCl,pH 7.6. MOPS medium was prepared (as described in Neidhardt, F. C., etal., J. Bacteriol., 119(3):736-747) (1974), which is hereby incorporatedby reference) as well as culture medium for enterobacteria. Tma endoVsonication buffer consisted of 20 mM HEPES, pH 7.4; 1 mM EDTA, pH 8.0;0.1 mM DTT; 0.15 mM PMSF; 50 mM NaCl. GeneScan stop solution consistedof 80% formamide (Amresco, Solon, Ohio), 50 mM EDTA (pH 8.0), 1% bluedextran (Sigma Chemicals). TB buffer (1×) consisted of 89 mM Tris and 89mM boric acid. TE buffer consisted of 10 mM Tris-HCl, pH 8.0 and 1 mMEDTA.

Proteinase K was purchased from QIAGEN (Valencia, Calif.). Microcon 30filters were purchased from Millipore (Bedford, Mass.). Taq DNApolymerase FS and four dideoxynucleotides were provided generously byPerkinElmer. Sep-Pak Cartridge C-18 was purchased from Waters (Milford,Mass.). Centri-Sep™ spin column P/N CS-90 was purchased from PrincetonSeparation (Adelphia, N.J.).

Thermus species AK16D DNA ligase was cloned, overexpressed in E coli.and purified to homogeneity as described in Tong, J., et al.,“Biochemical Properties of a High Fidelity DNA Ligase from ThermusSpecies AK16D,” Nucleic Acids Res 27:788-94 (1999), which is herebyincorporated by reference.

Example 2 Plasmid Construction, Cloning, Expression, and Purification ofThermotoga maritima Endonuclease V

Through BLAST searches (Altschul, S. F., et al., J. Mol. Biol.,215(3):403-10 (1990), which is hereby incorporated by reference), aputative open reading frame of 225 amino acid has been identified in theThermotoga maritima genome that shows 34% sequence identity to the E.coli endonuclease V gene. To prove that this, Tma ORF indeed encodes anendonuclease V, it was cloned and overexpressed in E. coli.

The putative endonuclease V gene (nfi) from Thermotoga maritima wasamplified by PCR using forward primer EV.Tma.01A (5′ GGA GGG AAT CAT ATGGAT TAC AGG CAG CTT CAC A 3′ (SEQ. ID. No. 3), the NdeI site isunderlined) and reverse primer EV.Tma.02R (5′ GCG CCT GGA TCC ACT AGTTCA GAA AAG GCC TTT TTT GAG CCG T 3′ (SEQ. ID. No. 4), the SpeI andBamHI sites are underlined). The PCR reaction mixture (100 μl) consistedof 50 ng of Thermotoga maritima genomic DNA, 10 μM of forward primerEV.Tma.01A, 10 μM of reverse primer EV.Tma.02R, 1× Pfu PCR buffer, 100μM of each dNTP, and 2.5 U Pfu DNA polymerase (Stratagene, La Jolla,Calif.). The PCR procedure included a pre-denaturation step at 95° C.for 2 min, 25 cycles of two-step amplification with each cycleconsisting of denaturation at 94° C. for 30 sec and annealing-extensionat 60° C. for 6 min, and a final extension step at 72° C. for 5 min. ThePCR product was purified by routine phenol extraction and ethanolprecipitation to remove thermostable Pfu DNA polymerase. Sambrook, J. etal., Molecular Cloning-A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), which is herebyincorporated by reference. The purified PCR product was digested withNdeI and BamHI, and ligated to pEV1 vector digested with the same pairof restriction enzymes. pEV1 is a derivative of pFBT69 (Barany, F.,Gene, 63:167-177 (1988), which is hereby incorporated by reference)which contains an NdeI site after the Shine-Dalgarno sequence in thephoA promoter region and a downstream multiple cloning site(BamHI-KasI-BstXI-EcoRV-EcoRI-MluI). The plasmid containing the putativeTma endonuclease V gene was designated as pEV5 and transformed into E.coli strain AK53 by a one-step protocol as described in Chung, C. T., etal., Proc. Natl. Acad. Sci. USA, 86(7):2172-75 (1989), which is herebyincorporated by reference. The endonuclease V gene is regulated by aphoA promoter. The insert containing Tma endonuclease V gene wassequenced to ensure authenticity of the plasmid constructs.

Overnight E. coli culture containing pEV5 was diluted 100-fold into FBmedium supplemented with 50 μg/ml ampicillin. The E. coli cells weregrown at 37° C. with 200 rpm shaking to an absorbance at 550 nm of 0.8.The cultures were diluted 50-fold into 1 liter of MOPS mediumsupplemented with 50 μg/ml ampicillin, and grown under the sameconditions overnight. After centrifugation at 3000×g for 10 min, thecell pellets were suspended in 10 ml of Tma endoV sonication buffer (20mM HEPES, pH 7.4; 1 mM EDTA, pH 8.0; 0.1 mM DTT; 0.15 mM PMSF; 50 mMNaCl) and sonicated on ice for two to three times at ten seconds each.

To purify the endonuclease V protein, cell debris were removed bycentrifugation at 3000×g for 15 min. The supernatants were incubated at70° C. for 15 min to denature thermolabile E. coli. host proteins. Afterseparating inactivated E. coli proteins by centrifugation at 10,000×gfor 20 min, the supernatants were dialyzed against the starting buffer(50 mM HEPES, pH 7.4, 1 mM EDTA, 50 mM NaCl, 0.1 mM DTT) overnight.

FIG. 2 shows a 12.5% SDS-PAGE gel demonstrating the purification ofThermotoga maritima endonuclease V protein at various stages in theprocess. Lane 2 shows the total proteins released from E. coli. cells.Since most E. coli host proteins are thermolabile, while Tma endoV isthermostable, the lysate was heated at 70° C. for 15 min in order toinactive most E. coli host proteins (including E. coli. endo V, SeeExample 4) while maintaining Tma endoV activity. After heat treatment,inactivated E. coli host proteins aggregate, and the majority of hostproteins can be pelleted and removed by centrifugation. This leaves TmaendoV as the major protein in the supernatant, as demonstrated in FIG. 2Lane 3 which shows proteins in the supernatant after heat treatment andcentrifugation. Tma endonuclease V eluted at about the 24 kDa position,indicating that it exists as monomer in solution (FIG. 2B).

Tma endonuclease V was purified to near homogeneity using a HiTrap SPcolumn containing sulphopropyl functional group (Pharmacia). In a bufferat pH 7.4, Tma endonuclease V carries a net positive charge, making it acompatible substrate for the HiTrap SP. The column was then eitherwashed stepwise with elution buffers containing increasing amounts ofNaCl, or washed with a buffer containing a NaCl concentration gradientin conjunction with an FPLC or HPLC system. Elution of a protein fromthe column is dependent on a particular NaCl concentration for thatprotein. In the stepwise elution, proteins were eluted with 150 mM NaClto 500 mM NaCl at 50 mM interval. Pure Tma EndoV was eluted with 250-300mM NaCl. FIG. 2, lane 4 demonstrates that the enzyme from the elutionwith 250-300 mM NaCl is nearly homogeneous. The concentration of theenzyme was then determined by the ultraviolet absorption method: proteinconcentration=A(280)×OD 280. Wetlaufer, D. B., Adv. Prot. Chem.17:303-390 (1962), which is hereby incorporated by reference. For TmaendoV, 1A=0.89 mg/ml, which was calculated by using the Protean softwareprogram (Power Macintosh version 3.05, DNA Star Inc.) and based on theprotein sequence of Tma endoV. The Tma endoV protein sequence (nfi gene)is available at the TIGR Microbial Database Locus TM1865 at web site:http://www.tigr.org/tdb/, under the section Thermotoga maritima.

To ensure that the purified Tma endonuclease V as shown in FIG. 2 isdevoid of endogenous E. coli endonuclease V which has a similarmolecular weight, the protein was transferred to a PVDF membrane andsubjected to N-terminal sequencing according to the procedure used inCao, W. et al., J. Biol. Chem., 273(49):33002-10 (1998), which is herebyincorporated by reference. The peptide sequencing result matched thepredicted N-terminal sequence of Tma nfi gene. Additionally, a 70° C.-15min heat-treatment was performed on E. coli endonuclease V purchasedfrom a commercial source (Trevigen, Gaithersburg, Md.). While theuntreated enzyme was active as reported, the heat-treated enzyme lostits enzymatic activity. Thus, the heating step used in purification islikely to have inactivated E. coli endonuclease V

Example 3 Oligonucleotide Substrates Preparation

E. coli endonuclease V demonstrates high activity with double-strandedDNA strands containing deoxyinosine-deoxyinosine or deoxyinosine-basemismatch. Yao, M. and Kow, Y. W., J. Biol. Chem., 269(50):31390-96(1994), which is hereby incorporated by reference. This generalcharacteristic of endoV was used in order to functionally identify thepurified Tma endoV enzyme. A double-stranded oligonucleotide containinga base mismatch was designed as a substrate to monitor for cleavageactivity by the purified enzyme. FIG. 3A shows a simple assay systemusing two differentially labeled fluorescent oligonucleotides. The topstrand is labeled with 6-FAM and the bottom strand is TET labeled. Themismatch position of the deoxyinosine nucleotide is off-center so thatnicked products do not comigrate on a denaturing polyacrylamide gel. Thedifferential double labeling allows the nicking events on both strandsto be easily observed and distinguished on the gel.

Oligonucleotide DNA substrates were purified on denaturing sequencinggels (7 M urea/10% polyacrylamide) as described in “The Complete Guide:Evaluating and Isolating Synthetic Oligonucleotides” (Applied BiosystemsInc., Foster City, Calif.)). Purified oligonucleotides were dissolved inTE buffer. Equal molar concentration of two complementary single strandswere mixed and incubated at 85° C. for 3 min and allowed to form duplexDNA substrates at room temperature for 30 min.

Example 4 Cleavage of Deoxyinosine-Containing Oligonucleotides

In order to functionally identify the purified enzyme,deoxyinosine-containing substrates were initially used since E. coliendonuclease V shows high activity toward an deoxyinosine-containingstrand. Yao, M. et al., J. Biol. Chem., 269(50):31390-96 (1994), whichis hereby incorporated by reference.

The cleavage reactions were performed at 65° C. for 30 minute in a 20 μlreaction mixture containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol,5 mM MgCl₂ unless otherwise specified, 10 nM DNA substrate, and theindicated amount of purified Tma endonuclease V protein. The reactionwas terminated by adding an equal volume of GeneScan stop solution. Thereaction mixtures were then heated at 94° C. for 2 min. and cooled onice. Three microliter samples were loaded onto a 10% GeneScan denaturingpolyacrylamide gel (Perkin Elmer). Electrophoresis was conducted at 1500voltage for 1 hr using an ABI 377 sequencer (Perkin Elmer). Cleavageproducts and remaining substrates were quantified using the GeneScananalysis software versions 2.1 or 3.0.

At a low enzyme concentration (E:S=1:10, S=10 nM, E:S stands for theratio of enzyme-to-substrate, S stands for substrate), Tma endonucleaseV nicked exclusively at deoxyinosine-containing strands regardless ofwhether deoxyinosine was placed on the top or bottom strand or both(FIG. 3B). Cleavage was efficient for all four kinds of deoxyinosinebase-pair (I/A, I/G, I/C, I/T), confirming a previous observation withthe E. coli enzyme. Yao, M. et al., J. Biol. Chem., 269(50):31390-96(1994), which is hereby incorporated by reference. The predominantproducts were formed by nicking at the 3′ side one nucleotide after theinosine base on both strands (FIG. 3D). At a high enzyme concentration(E:S=10:1, S=10 nM), Tma endonuclease V nicked deoxyinosine-containingstrands effectively, resulting in virtually complete conversion tonicked products (FIG. 3C). In contrast to the results obtained with thelow enzyme concentration (FIG. 3B), the high enzyme concentrationpromoted opposite strand nicking (e.g., nicking the A-containing strandin a dI/dA duplex oligonucleotide substrate). For I/A and I/Gsubstrates, the nicking primarily occurred at the 3′ side one nucleotidebeyond the mismatched A or G base. However, opposite strand nicking forI/C and I/T substrates generated an additional product at a lowermolecular weight position (FIG. 3C). Comparison with length markerssuggested that the cleavage site was approximately 2-3 nt at the 5′ sideof the T or C base as the 38 mer and 27 mer represent cleavage productsright after the T or C base at the 3′ side (FIG. 3D). The oppositestrand nicking was incomplete at I/C and I/T, while the deoxyinosinecleavage was complete, indicating that the 5′ nicking of C or T hadoccurred after nicking of the deoxyinosine-containing strand. It isunknown whether the high yield opposite strand nicking at the 5′ side inI/C is associated with the fact that I/C forms a Watson-Crick base-pair(Xuan, J. C. et al., Nucleic Acids Res., 20:5457-64 (1992), which ishereby incorporated by reference) and whether the low yield oppositestrand nicking at the 5′ side in I/A and I/G is associated with the factthat they form non-Watson-Crick base-pairs. Corfield, P. W., et al.,Nucleic Acids Res., 15(19):7935-49 (1987), which is hereby incorporatedby reference. The opposite strand nicking at the 5′ side of the I/C andI/T base-pairs was not observed with C/I or T/I substrates, indicatingthat either the nicking products were less stable at 65° C. incubationand being degraded as single-stranded DNA or the nicking at thesesubstrates were less efficient due to sequence context. These resultssuggest that nicking events in general occur at the 3′ side, but theenzyme is capable of cleaving at the 5′ side at the opposite strandcomplementary of an deoxyinosine-containing strand. An earlier studysuggested that E. coli endonuclease V cleaves at the 5′ side ofmethylbenz[a]anthracene adducts. Demple, B. et al., J. Biol. Chem.,257(6):2848-55 (1982), which is hereby incorporated by reference. Thus,the enzyme is able to make a 5′ incision at some lesion sites. The aboveresults confirmed that the purified enzyme is an endonuclease V.

Example 5 Effect of Reaction Buffer on Tma Endonuclease V Activity andSpecificity

Since enzyme activity and specificity can be influenced by the metalcofactor used by an enzyme, Tma endoV mismatch cleavage activity in thepresence of either Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺,Ba²⁺ or Sr²⁺ was analyzed. The concentration of each metal was 5 mM. Thereaction mixture contained 10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol,5 mM metal chloride, 100 nM Tma endoV and 10 nM oligonucleotidescontaining base-mismatch either (A/A), or (C/T), or (C/C), a regularoligonucleotide without base mismatch was used as a control. The mixturewas incubated at 65° C. for 30 min and loaded on a gel forelectrophoresis. Cleavage products and remaining substrates werequantified using the GeneScan analysis software versions 2.1 or 3.0.Cleavage products were only observed in the presence of Mg²⁺ or Mn²⁺,indicating that these two cations are the only metal cofactors of Tmaendo V in the set tested. FIG. 4 shows Tma endo V activity in thepresence of different concentrations of Mg²⁺ or Mn²⁺ usingdouble-stranded oligonucleotide DNA with an A/A mismatch as substrate.The mismatch cleavage activity was most active at Mg²⁺ concentrations of2-7 mM and at Mn²⁺ concentrations of 0.4-1.2 mM.

Example 6 Cleavage of Base Mismatch-Containing Oligonucleotides

Base mismatch nicking was investigated using 12 substrates containingdifferent base mismatches (FIG. 5). The cleavage reactions wereperformed at 65° C. for 30 minute in a 20 μl reaction mixture containing10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 5 mM MgCl₂ or 1 mM MnCl₂,10 nM DNA substrate and the indicated amount of purified Tmaendonuclease V protein. The reaction was terminated by adding an equalvolume of GeneScan stop solution. The reaction mixtures were then heatedat 94° C. for 2 min. and cooled on ice. 3 μL of samples were loaded ontoa 10% GeneScan denaturing polyacrylamide gel (Perkin Elmer).Electrophoresis was conducted at 1500 voltage for 1 hr using an ABI 377sequencer (Perkin Elmer). Cleavage products and remaining substrateswere quantified using the GeneScan analysis software versions 2.1 or3.0. With Mg²⁺ as the metal cofactor, the enzyme needed a higherenzyme-to-substrate (E:S) ratio to make an incision at a mismatch (FIG.5A-5B). With Mn²⁺ as the metal cofactor, the nicks at mismatchsubstrates occurred at lower E:S ratios (FIG. 5C-5D). The cleavagereactions of mismatches did not proceed to completion, as even at higherE:S ratios there were still significant remaining mismatch containingsubstrates while the nonspecific nicking became significant (FIG. 5B).The cleavage sites were identical to those identified usingdeoxyinosine-containing substrates, i.e., at the 3′ side one nucleotideafter the mismatches. The mismatch nicking was most efficient with an Aor G base, and less so with a T base. C base was nicked much lessefficiently when it base-pairs with an A or a T base, and cleavage wasnot observed when it base-pairs with a C. This mismatch cleavage profileis in agreement with the E. coli enzyme. Yao, M. et al., J. Biol. Chem.,269(50):31390-96 (1994), which is hereby incorporated by reference.However, no strand-preferred or terminus-dependent mismatch cleavage wasobserved as previously reported using the E. coli enzyme (Yao, M. etal., J. Biol. Chem., 269(50):31390-96 (1994), which is herebyincorporated by reference). Base mismatch cleavage occurred at both topand bottom strands regardless of the distance between the mismatch baseand the 5′ termini (FIG. 2A, FIG. 5A). The finding that Tma EndoV wascapable of cleavage of both top and bottom strands regardless of thedistance between the mismatch base and the 5′ termini was unanticipatedand demonstrates that this enzyme has unique properties whichdistinguish it from the E. coli enzyme. The same authors whocharacterized the mesophilic E. coli EndoV enzyme most recentlycharacterized the thermostable A. fulgidus EndoV enzyme (Liu et. al.,Mutation Research 461:169-177 (2000). The A. fulgidus EndoV enzyme onlyhad activity against inosine containing DNA, not against mismatches orother lesions. Thus, it cannot be presumed that a thermostable EndoVwould have activity on substrates containing base mismatches.

Table 1 provides a summary of results with Tma endonuclease V cleavageof heteroduplexed synthetic substrates containing single basemismatches. Note that for every possible base change, there are twopossible heteroduplexed products which may form: A

G (A-C, G-T); C

T (C-A, T-G); A

C (A-G, C-T); G

T (G-A, T-C); A

T (A-A, T-T), and G

C (G-G, C-C). TABLE 1 Summary of Tma endonuclease V cleavage ofheteroduplexed synthetic substrates containing single base mismatches.Base change (Wt

Mt) A

G C

T A

C G

T A

T G

C Heteroduplex I: UpperStrand (Wt) A +++ C + A ++ G ++ A +++ G +++ | | || | | BottomStrand (Mt) C − A ++ G +++ A ++ A +++ G +++ Heteroduplex II:UpperStrand (Mt) G ++ T ++ C + T ++ T + C − | | | | | | BottomStrand(Wt) T ++ G +++ T ++ C − T + C −Note:UpperStrand:5′-FAM-TA CCC CAG CGT CTG CGG TGT TGC GTN AGT TGT CAT AGT TTG ATC CTCTAG TCT TGT TGC GGG TTCC-3′ (SEQ. ID. No. 5)BottomStrand:3′- GGG GTC GCA GAC GCC ACA ACG CAN TCA ACA GTA TCA AAC TAG GAG ATC AGAACA ACG CCC-TET-5′ (SEQ. ID. No. 6)Cleavage symbols:(+++): high intensity cleavage. (++): intermediate intensity cleavage.(+): low intensity cleavage. (−) no cleavageThe cleavage of these heteroduplexed products are not always identical(i.e. compare A-C with C-A), and this reflects subtleties in thestructure of the DNA as a consequence of neighboring sequence variation.Nevertheless, for each possible base change, signal is generated for atleast one top strand and at least one bottom strand. Thus, the Tma EndoVenzyme should be able to recognize any possible single base mutation orpolymorphism.

Example 7 Non-Specific Cleavage Activity of Tma Endonuclease V on SingleStranded and Double Stranded DNA

In order to determine the non-specific cleavage activity of Tma endoV,the cleavage activities on a single strand oligonucleotide and a regularplasmid were measured. The single strand DNA cleavage reactions wereperformed at 65° C. for 30 minute in a 20 μl reaction mixture containing10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, in 5 mM MgCl₂ or 0.6 mMMnCl₂, 10 nM single strand DNA substrate, and the indicated amount ofpurified Tma endonuclease V protein.

The plasmid cleavage reactions were performed at 65° C. for 30 minute ina 20 μl reaction mixture containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2%glycerol, 5 mM MgCl₂ or 1 mM MnCl₂, 10 nM plasmid pFB 7.6, and theindicated amount of purified Tma endonuclease V protein.

The reaction was terminated by adding an equal volume of GeneScan stopsolution. The reaction mixtures were then heated at 94° C. for 2 min.and cooled on ice. Three microliter of samples were loaded onto a 10%GeneScan denaturing polyacrylamide gel (Perkin Elmer). Electrophoresiswas conducted at 1500 voltage for 1 hr using an ABI 377 sequencer(Perkin Elmer). Cleavage products and remaining substrates quantifiedusing the GeneScan analysis software versions 2.1 or 3.0.

The enzyme nicked the AP site or uracil-containing strand specificallyat low enzyme concentrations (FIG. 6A, lanes 3-4 and 6-7). When theenzyme concentration was increased to 100 nM (E:S=10:1), opposite strandnicking started to occur (FIG. 6A), lanes 5 and 8), suggesting thatopposite strand nicking is not unique to inosine-containing substrates.The enzyme formed a weak but distinct complex with an AP site substrate,but not with a uracil substrate (FIG. 6B), suggesting that the enzymedoes not solely rely on base recognition for achieving ground statebinding. The lack of stable binding to a uracil site is consistent withgenetic studies which indicate that endoV does not play a significantrole in uracil repair. Guo, G. et al., J. Bacteriol, 180:46-51 (1998),which is hereby incorporated by reference.

The binding data obtained from I/I substrate showed that the enzyme maybe able to interact with inosine substrate in a single-stranded fashion.Previous studies demonstrate that E. coli endoV cleaves single-strandedinosine substrate (Yao, et al., J. Biol. Chem., 271:30672-76 (1996),which is hereby incorporated by reference). To gain a betterunderstanding of how the enzyme cleaves single stranded DNA, cleavageand binding of inosine, AP site, and uracil substrates were examined.Tma endoV cleaved single stranded inosine substrate with either Mg²⁺ orMn²⁺ as the metal cofactor (FIG. 6C). The cleavage of single stranded APsite or uracil appeared to prefer using Mn²⁺ as the metal factor (FIG.6C). In addition, Mn²⁺ promoted the cleavage of non-specific singlestranded DNA (FIG. 6C). The nonspecific endonuclease activity wasfurther confirmed using a supercoiled plasmid substrate (FIG. 6D). WithMg²⁺ as the metal cofactor, the non-specific endonuclease activityprimarily nicked the plasmid once. With Mn²⁺ as the metal cofactor, theenzyme could nick a plasmid molecule at least twice to generate a linearplasmid (FIG. 6D). The two nicking events are sequential as evidenced bythe appearance of nicked plasmid intermediate. The conversion ofsupercoiled plasmid into nicked or linear plasmid suggests that theenzyme does not need free 5′ or 3′ ends to access a DNA molecule.

As with the double stranded inosine substrate, binding tosingle-stranded inosine substrate also requires a metal cofactor (FIG.6E). The binding affinity to the single-stranded inosine substrateappeared to be weaker than the double-stranded (FIG. 6E). A stablecomplex was formed in the presence of Mg²⁺, suggesting that Tma endoVmaintained a relatively high affinity to the nicked single-strandedproduct (FIG. 6F). Single-stranded inosine cleavage activity may helprepair damage at transient single-stranded regions during replicationand transcription.

This indicates that nonspecific cleavage is more prevalent with Mn²⁺ asthe metal cofactor. In addition, the conversion of a supercoiled plasmidinto nicked and linear forms suggests that the enzyme does not need free5′ or 3′ end to access a DNA molecule. Since the above resultsdemonstrate that Tma endoV cleavage is more specific in the presence ofMg²⁺ than Mn²⁺, Mg²⁺ was determined to be the preferred metal cofactorin the reaction buffer.

Example 8 The Effects of pH on the Base-Mismatch Cleavage Activity ofTma Endonuclease V

pH can exert a profound effect on mismatch cleavage. The substrates inthis assay are a group of four oligonucleotides, three of them containmismatches (A/A), (C/T), and (C/C) respectively, the other is a regularoligonucleotide without mismatch. The cleavage reactions performed at65° C. for 1 hour in the buffer containing 10 mM HEPES (pH 7.4), 1 mMDTT, 2% glycerol, 5 mM MgCl₂ (or 1 mM MnCl₂), 10 nM DNA substrate,indicated that the amount of purified Tma endonuclease V protein withdifferent E:S ratios.

The following buffers were used in making different pH: 20 mM MES (pH6.0), 20 mM MOPS (pH 6.5), 10 mM HEPES (pH 7.0-7.5), 20 mM Tris (pH8.0-8.5). A. Reactions performed with 100 nM endonuclease V (E:S=10:1)in the presence of 5 mM MgCl₂. B. Reactions performed with 10 nMendonuclease V (E:S=1:1) in the presence of 1 mM MnCl₂.

The reaction mixtures were then heated at 94° C. for 2 min. and cooledon ice. Three microliter samples were loaded onto a 10% GeneScandenaturing polyacrylamide gel (Perkin Elmer). Electrophoresis wasconducted at 1500 voltage for 1 hr using an ABI 377 sequencer (PerkinElmer). Cleavage products and remaining substrates were quantified usingthe GeneScan analysis software versions 2.1 or 3.0.

FIG. 7 shows that the pH of the reaction buffer can exert profoundeffects on mismatch cleavage. Nicks at the mismatch position becameobservable at pH 6.5 and reached a maximum at pH 7.5 with concurrentincreases in nonspecific cleavage both in the presence of either 5 mMMg²⁺ (E:S=10:1), FIG. 7A, or 1 mM Mn²⁺ (E:S=1:1) FIG. 7B. Consistentwith previous studies for E. coli endonuclease V, (Gates, F. T., 3rd andLinn, S., J. Biol. Chem., 252(5):1647-53 (1977), which is herebyincorporated by reference) and E. coli DNA polymerase (Eckert, K. A., etal., “Effect of Reaction pH on the Fidelity and Processivity ofExonuclease-Deficient Klenow Polymerase” J. Biol. Chem.,268(18):13462-71 (1993), which is hereby incorporated by reference). Tmaendonuclease V thus became more nonspecific at high pH conditions.

Example 9 The Effects of Salt on the Cleavage of OligonucleotidesContaining a Base Mismatch

To study how reaction conditions may affect base mismatch cleavage,representative mismatch cleavage was tested at different NaClconcentrations. The substrates in this assay were a group of fouroligonucleotides, three of them containing (A/A), (C/T), and (C/C)mismatches, respectively, while the other was a regular oligonucleotidewithout mismatch. The cleavage reactions were performed at 65° C. for 30minute in a 20 μl reaction mixture containing 10 mM HEPES (pH 7.4), 1 mMDTT, 2% glycerol, 5 mM MgCl₂ or 1 mM MnCl₂, 10 nM DNA substrate, and theindicated amount of purified Tma endonuclease V protein. Theconcentration of NaCl ranged from 0-250 mM with 50 mM intervals. Thereaction was terminated by adding an equal volume of GeneScan stopsolution. The reaction mixtures were then heated at 94° C. for 2 min.and cooled on ice. 3 μL of samples were loaded onto a 10% GeneScandenaturing polyacrylamide gel (Perkin Elmer). Electrophoresis wasconducted at 1500 voltage for 1 hr using an ABI 377 Sequencer (PerkinElmer). Cleavage products and remaining substrates were quantified usingthe GeneScan analysis software versions 2.1 or 3.0.

It was apparent that the enzyme preferred a low salt environment formismatch cleavage with either Mg²⁺ or Mn²⁺. With Mg²⁺ as the metalcofactor, the mismatch cleavage yields were highest without salt. WithMn²⁺ as the metal cofactor, the mismatch cleavage yields were highestwith 50 mM NaCl. See FIG. 8. This is in agreement with the notion thatincreased salt concentrations reduce DNA binding affinity. No cleavagewas observed with either the C-C mismatch or A-T match under theseconditions.

From the above experiments, an optimal reaction buffer condition inwhich Tma endo V mismatch cleavage is maximized and nonspecific mismatchcleavage is minimized was determined. The optimal conditions are 10 mMHEPES (pH 7.4), 1 mM DTT, 2% glycerol, and 5 mM MgCl₂. These conditionswere determined from data primarily using oligonucleotides as substrate.Therefore, the optimal condition may vary when different substrates,such as genomic DNA, are utilized. In addition, this optimal conditionis one that appears best with respect to cleavage characteristics mostdesirable in the context of the present invention, but it should benoted that sub-optimal conditions are also compatible in the context ofthe invention.

Example 10 Optimization of Mg²⁺ and Mn²⁺ Concentration in TmaEndonuclease V Cleavage Reaction Condition when SyntheticOligonucleotide DNA was Used as Substrate

Using fluorescences labeled double strand oligonucleotide containing(A/A) mismatch as the substrate, the cleavage activity of Tma endoV wasmeasured in different concentrations of Mg²⁺ or Mn²⁺. The cleavagereactions were performed at 65° C. for 30 minute in a 20 μl reactionmixture containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, unlessotherwise specified, 10 nM DNA substrate, different concentrations ofMg²⁺ or Mn²⁺, and 100 nM Tma endoV (in the presence of Mg²⁺) or 10 nMTma endoV (in the presence of Mn²⁺).

The reaction was terminated by adding an equal volume of GeneScan stopsolution (50 mM EDTA, 80% formamide, 1% blue dextran). The reactionmixtures were then heated at 94° C. for 2 min. and cooled on ice. 3 μLof samples were loaded onto a 10% GeneScan denaturing polyacrylamide gel(Perkin Elmer). Electrophoresis was conducted at 1500 voltage for 1 hrin an ABI 377 sequencer (Perkin Elmer). Cleavage products and remainingsubstrates were quantified using the GeneScan analysis software versions2.1 or 3.0 (Perkin Elmer). The quantitative results are shown in FIG. 4.The optimal concentration for Mg²⁺ is 2-7 mM and for Mn²⁺ is 0.4-1.2 mMin the cleavage of DNA substrate containing a base mismatch.

Example 11 PCR Amplification

Genomic DNA containing mutations in k-ras gene codon 12 and 13 wereextracted from cell lines containing the mutations. Ccell line ht 29 orsw1417 contains normal genomic DNA. Cell line sw620 or sw480 has DNAcontaining pure g12v(g→t) mutation. The ratio of wild type-to-mutantg12d(g→a) in the genomic DNA extracted from cell line ls180 is 1:1.8.The ratio of wild type-to-mutant g12a(g→c) in the DNA extracted fromcell line sw1116 is 1:0.7. The ratio of wild type-to-mutant g13d(g→a) inthe genomic DNA extracted from cell line hct 15 or dld 1 is 1:1.1.

In carrying out the PCR amplification process, forward and reverse PCRprimers can be synthesized with 5′ end labels of TET and 6-FAM(PE-Biosystems, Foster City, Calif.). These two fluorescent groupsappear green and blue, respectively, when analyzed by an ABI 377 DNAsequencer. Differential labeling of the top and bottom strands allowsone to distinguish cleavage products from each strand independently. Tominimize non-specific cleavage of TET and 6-FAM labels by Tma endoV,three additional cytosine deoxynucleotides were synthesized on the 5′end of each primer. Examples of suitable primers are shown in Table 1A.TABLE 2A PCR Primer Sequences for Amplifying Cancer Gene Fragments. GeneExon Sequence K-ras Exon 1 TopTet-5′-CCCCATAGTGTATTAACCTTATGTGTGACATGTTC-3′ (SEQ. ID. No. 7) BottomFam 5′-CCCCAAAATGGTCAGAGAAACCTTTATCTGTATC-3′ (SEQ. ID. No. 8) APC Exon15 Top Tel-5′-CCCCGCTGCCACTTGCAAAGTTTCTTC-3′ (SEQ. ID. No. 9) BottomFam-5′-CCCCACTCTGAACGGAGCTGGCAAT-3′ (SEQ. ID. No. 10) Exon 5 TopTet-5′-CCCCTGTTCACTTGTGCCCTGACTTTC-3′ (SEQ. ID. No. 11) BottomFam-5′-CCCCCAGCTGCTCACCATCGCTATC-3′ (SEQ. ID. No. 12) Exon 6 TopTet-5′-CCCCCTCTGATTCCTCACTGATTGCTCTTA-3′ (SEQ. ID. No. 13) BottomFam-5′-CCCGGCCACTGACAACCACCCTTAAC-3′ (SEQ. ID. No. 14) p53 Exon 7 TopTet-5′-CCCGCCTCATCTTGGGCCTGTGTTATC-3′ (SEQ. ID. No. 15) BottomFam-5′-CCCGTGGATGGGTAGTAGTATGGAAGAAAT-3′ (SEQ. ID. No. 16) Exon 8 TopTet-5′-CCCGGACAGGTAGGACCTGATTTCCTTAC-3′ (SEQ. ID. No. 17) BottomFam-5′-CCCCGCTTCTTGTCCTGCTTGCTTAC-3′ (SEQ. ID. No. 18) 1.7kb TopFam-5′-CCCGCATGGTGGTGCACACCTATAGTC-3′ (SEQ. ID. No. 19) BottomTet-5′-CCCAAGCTGTTCCGTCCCAGTAGATTAC-3′ (SEQ. ID. No. 20) BRCA 1 Exon 2Top Tet-5′-CCCCTCATTGGAACAGAAAGAAATGGATTTATC-3′ (SEQ. ID. No. 21) BottomFam-5′-CCCCTCTTCCCTAGTATGTAAGGTCAATTCTGTTC-3′ (SEQ. ID. No. 22) Exon 20Top Tet-5′-CCCCACTTCCATTGAAGGAAGCTTCTCTTTC-3′ (SEQ. ID. No. 23) BottomFam-5′-CCCCATCTCTGCAAAGGGGAGTGGAATAC-3′ (SEQ. ID. No. 24) BRCA 2 Exon 11Top Tet-5′-CCCCCAAAATATGTCTGGATTGGAGAAAGTTTC-3′ (SEQ. ID. No. 25) BottomFam-5′-CCCCTTGGAAAAGACTTGCTTGGTACTATCTTC-3′ (SEQ. ID. No. 26) VHL Exon 1Top Tet-CCCGACCGCGCGCGAAGACTAC-3′ (SEQ. ID. No. 27) BottomFam-5′-CCCAGGGGCTTCAGACCGTGCTATC-3′ (SEQ. ID. No. 28) Exon 2 TopTet-5′-CCCCACCGGTGTGGCTCTTTAACAAC-3′ (SEQ. ID. No. 29) BottomFAM-5′-CCCCTGACATCAGGCAAAAATTGAGAA-3′ (SEQ. ID. No. 30) Exon 3 TopTet-5′-CCCTAGTTGTTGGCAAAGCCTCTTGTTC-3′ (SEQ. ID. No. 31) BottomFam-5′-CCCAAACTAAGGAAGGAACCAGTCCTGTATC-3′ (SEQ. ID. No. 32)

In the process of purification of PCR primers, 200 ng of labeled primerswas dissolved in 20 μl of ddH₂O and was mixed with an equal volume offormamide. After incubation at about 64° C. for 2 min, the primers wereloaded on a 10% polyacrylamide gel containing 7 M urea. Afterelectrophoresis, the gel slices containing pure primers were cut out andsoaked in TNE solution (0.1 M Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM EDTA)at 37° C. overnight. The solution containing gel slices was removed andloaded on a Sep-Pak Cartridge C-18 (Waters, Milford, Mass.) pre-washedwith methanol and water. After washing with 20 ml of ddH₂O, the primerswere eluted out with 2 ml of elution buffer (5 mM TEAA (triethylamineacetate), 50% methanol) and dried with a speed vacuum. The pellets weresuspended with TE buffer. PCR reactions were performed in a GeneAmp PCRSystem 2400 or GeneAmp PCR System 9700. 50 μl of PCR reaction solutioncontains 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM of each dNTP, 0.2 μMof primers, 2.5 mM MgCl₂, and about 100 ng genomic DNA or amplicons. Inthe amplification of VHL exon 1 with high GC content, 2% DMSO wasincluded in the PCR reaction mixture.

PCR amplification may be directly from genomic DNA or, alternatively,from amplicon(s) containing the fragment(s) of interest. Thermocyclingconditions for the PCR reaction should be adjusted to reduce theamplification of non-specific fragments. Table 2B shows typicalthermocycling conditions for various genes. TABLE 2B PCR CyclingConditions for Amplifying Fragments in Different Genes. DNA InitialCycle reaction Final Gene Exon polymerase denature cycle number reactionextension K-ras Exon 1 AmpliTaq 94° C. 30 94° C. for 15 sec, 72° C. for7 min for 2 min 60° C. for 2 min APC Exon 15 Gold Taq 95° C. 30 94° C.for 30 sec 72° C. for 7 min for 10 min 63° C. for 1 min 15 sec p53 Exon5 AmpliTaq 95° C. 35 94° C. for 20 sec 72° C. for 7 min Exon 6 for 2 min65° C. for 2 min Exon 7 Gold Taq 95° C. 35 94° C. for 30 sec 72° C. for7 min for 10 min 60° C. for 30 sec 72° C. for 1 min Exon 8 AmpliTaq 95°C. 35 94° C. for 20 sec 72° C. for 7 min for 2 min 65° C. for 2 min 1.7kb TaqPlus 95° C. 35 94° C. for 20 sec 72° C. for 7 min precision for 2min 68° C. for 2 min 45 sec DNA polymerase mixture BRCA 1 Exon 2 GoldTaq 95° C. 35 94° C. for 30 sec 72° C. for 7 min Exon 20 for 10 min 60°C. for 30 sec BRCA 2 Exon 11 72° C. for 1 min VHL Exon 1 AmpliTaq 95° C.35 94° C. for 20 sec 72° C. for 7 min for 2 min 66° C. for 30 sec 72° C.for 1 min Exon 2 AmpliTaq 95° C. 35 94° C. for 20 sec 72° C. for 7 minfor 2 min 60° C. for 30 sec 72° C. for 1 min Exon 3 AmpliTaq 95° C. 3594° C. for 20 sec 72° C. for 7 min for 2 min 66° C. for 30 sec 72° C.for 1 minNote:for Amplitaq and Taqplus DNA polymerase, after initial denature step,DNA polymerase was added to perform hot start PCR. For gold Taq DNApolymerase, DNA polymerase was included in the reaction mixture beforePCR reaction starts. The PCR machine is GeneAmp PCR system 2400 or 9700(Perkin Elmer).

Example 12 Preparation of Heteroduplex DNA Substrates

PCR fragments were incubated with proteinase K (20 mg/ml, QIAGEN) in aratio of 1 μl of proteinase K to 12 μl of PCR products to remove Taq DNApolymerase. The reaction was carried out at 70° C. for 10 min and at 80°C. for 10 min to inactivate the Proteinase K. The mixture was heated at94° C. for 1 min, 65° C. for 15 min and then cooled down to roomtemperature to form heteroduplex DNA. For genomic DNA from cell linescontaining more mutant than normal DNA, such as cell line LS180, PCRfragments from normal genomic DNA were added to make the final ratio(mutant-to-wild type) 1:1. The ratio of mutant to wild type in cell lineSW1116 is 0.7:1.0, and the pure mutant DNA is not available, so thegenomic DNA alone was treated as described in the following text. If thegenomic DNA is purified from blood, for example, where the target DNA isa mutation in the APC, BRCA 1, BRCA 2 and VHL genes, the PCR fragmentswere treated as described before without adding wild-type PCR fragments.

Example 13 Optimization of Conditions of Mutation Detection by TmaendoV/Ligase when Using Heteroduplex PCR Fragments as Substrate

When synthetic double stranded oligonucleotides (60-66 mer) were used asthe DNA substrate for endoV cleavage, the optimal buffer condition is 10mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 5 mM MgCl₂ in which Tma endoVhas high specific cleavage activity and low non-specific cleavageactivity. However, when an approximately 300 bp PCR fragment containingthe G12V mutation in k-ras gene was used as the substrate, cleavage wasnot observed. For this Tma endo V cleavage assay, a variety ofheteroduplex PCR fragments containing K-ras codon 12 mutation were usedas substrates. In these experiments, a mixture of wild-type and G12Vmutant DNA PCR amplified fragments were used. The two resultantheteroduplexed fragments contain G-A and T-C mismatches, respectively.Cleavage of both top and bottom strand were observed, products wereseparated on an ABI 377 DNA sequencing apparatus, and amount of cleavageproduct was quantified using GeneScan 3.0 analysis software(PE-Biosystems, Foster City, Calif.). FIG. 9 demonstrates that adding 5%DMSO to the reaction will enhance mismatch cleavage activityapproximately 2-3 fold. Other organic solvents which may enhancemismatch cleavage include dimethyl formamide, ethylene glycol, glycerol,formamide, etc.

A second approach to improving cleavage involved the addition ofbetaine, N,N,N-trimethylglycine. Betaine was initially used to equalizethe melting temperatures of DNA fragments having different GC content.It was found that in certain “isostabilizing” concentrations of betaine,AT and GC pairs are equally stable. Rees, W. A., et al., Biochemistry,32:137-44 (1993), which is hereby incorporated by reference. Betaine wasalso used as an additive to facilitate PCR amplification of regions withhigh GC content. Henke, W. et al., “Betaine Improves the PCRAmplification of GC-Rich DNA Sequences,” Nucleic Acids Res., 25:3957-58(1997), which is hereby incorporated by reference.

FIG. 10 shows that the addition of betaine to the reaction buffer canfacilitate the base mismatch cleavage activity of Tma. Endo V, but itdoes so in a fragment dependent fashion. For PCR fragments with a low GCcontent, such as ones generated from the APC gene, the cleavage activityreached a maximum of 2 fold stimulation at 1.0 M betaine. For PCRfragments with a high GC content, for example the K-ras fragments,maximum cleavage activity was observed at 1.5 M betaine and resulted ina greater stimulation of 2.7 fold. The relative stimulation for bothhigh and low GC content fragments was approximately the same, 2 fold, at1 M betaine.

The effect of NaCl was also re-examined for heteroduplex fragments, andFIG. 11A demonstrates that addition of NaCl inhibits the cleavagereaction. At 50 mM NaCl, the cleavage reaction is almost completelyinhibited. FIG. 11B shows the effect of KCl on the mismatch cleavageactivity of Tma endoV. For this assay 100 ng of heteroduplex PCRfragments containing the K-ras G12V (G→T) mutation was used assubstrate, and the reaction was carried out at 65° C. for 1 hour in amodified optimal reaction buffer (2.0 M betaine, 10% DMSO) with 100 nMTma endonuclease V and varying concentrations of KCl. The results showthat the effect of KCl is indeed similar to that of NaCl, and that inthe presence of 50 mM KCl, Tma endoV cleavage activity is almostcompletely inhibited. Therefore, in the ligation step of this invention,the Tma endoV cleavage activity is essentially eliminated. These resultsconfirm that no additional salt should be included in the reactionbuffer.

The above analysis suggests that when PCR fragments are used assubstrates, the addition of 5% DMSO and 1-1.5 M betaine to the reactioncan significantly enhance specific cleavage activity of Tma endo V,while the addition of salt should be avoided.

Using the new optimal reaction conditions, a time course of the Tma endoV cleavage reaction was performed at 65° C. using PCR fragmentscontaining the K-ras G12D(G→A) mutation (FIG. 12). In these experiments,a mixture of wild-type and G12D mutant DNA PCR amplified fragments wereused. The two resultant heteroduplexed fragments contain G-T and A-Cmismatches, respectively. Cleavage of both top and bottom strand wereobserved, products were separated on an ABI 377 DNA sequencingapparatus, and the amount of cleavage product was quantified usingGeneScan 3.0 analysis software. The products increased linearly over thehour time course. Longer incubations can lead to an undesirable increaseof nonspecific products. Therefore, the optimal reaction conditions forTma endoV using heteroduplex PCR fragments as the substrate, in areaction buffer containing 10 mM HEPES (pH 7.4), 5 mM MgCl₂, 1 mM DTT,2% glycerol, approx. 1-1.5 M betaine, and 5% DMSO, and an incubation at65° C. for 1 h.

FIG. 13 (-DNA Ligase Lanes) demonstrates that the new optimal reactionconditions result in high activity and relatively good specificity ofthe Tma endo V enzyme when PCR fragments are used as substrates.Nevertheless, non-specific nicking is still observed, and as a resultthere exists significant background signal.

Example 14 Ligation Reaction Condition with NAD⁺ DNA Ligase

The optimal reaction buffer for Tsp.AK16D ligase is 20 mM Tris-HCl pH8.5, 5 mM MgCl₂, 50 mM KCl, 10 mM DTT, 1 mM NAD⁺, 20 μg/ml BSA. Tong,J., et al., “Biochemical Properties of a High Fidelity DNA Ligase fromThermus Species AK16D,” Nucleic Acids Res., 27:788-94 (1999), which ishereby incorporated by reference. Fifteen μL of the reaction mixturefrom the EndoV cleavage were added to 2 μl of 10× supplemental buffer(200 mM Tris-HCl, pH 8.5, 12.5 mM MgCl₂, 500 mM KCl, 100 mM DTT and 200μg/ml BSA), 1 μl of 20 mM NAD⁺, and 2 μl of 10-60 nM Tsp. AK16D DNAligase. The final concentration of the mixture is: 20 mM Tris-HCl pH8.5, 5 mM MgCl₂, 50 mM KCl, 10 mM DTT, 1 mM NAD⁺, 20 μg/ml BSA and 1-6nM AK16D DNA ligase. The mixture was incubated at 65° C. for 20 min andterminated by adding an equal volume of GeneScan stop solution (50 mMEDTA, 1% blue dextran and 80% formamide). The reaction mixtures werethen heated at 94° C. for 2 min. and cooled on ice. 2-3 μl of themixture were loaded onto a 6% denaturing polyacrylamide gel andelectrophoresed for 1 hr in an ABI 377 sequencer (Perkin Elmer) underthe following conditions: voltage of 1000 volts, current of 60 mA, powerof 200 w, and gel temperature of 45° C. 6-FAM and TET were thefluorescent group labelled in top and bottom strand primers,respectively. They appear blue and green, respectively, with an ABI DNAsequencer 377. Therefore, it can be concluded that the TET labeled(green) cleavage bands were generated from the top strand and FAMlabeled (blue) cleavage bands were from the bottom strand. Since TAMRAlabeled GeneScan Molecular size standard 500 (red bands) was loaded onthe same gel, the molecular weight of cleavage products could beestimated by comparison of the mobility of the size standards to thecleavage products using the GeneScan analysis software versions 2.1 or3.0a.

Example 15 Detection of Codon 12 and 13 Mutations in K-ras Gene

The strategy of mutation scanning with Tma endoV/AK16D DNA ligase wasfirst tested in the detection of codon 12 and 13 mutations in the K-rasgene. Genomic DNA from cell lines containing mutations in codon 12 and13 were used as templates for PCR amplification. The cell linescontaining K-ras mutation are listed in Table 3 as follows: TABLE 3Ratio of Cell Line Genotype mutant-to-wild type HT29 Wild type SW1417SW1116 G12A (G→C) 0.7:1 DLD1 G13D (G→A)   1:1 HCT 15 LS180 G12D (G→A)1.8:1 SW620 Pure G12V (G→T) Sw480The sequence of top strand PCR primer was:Tet-5′-CCCCATAGTGTATTAACCTTATGTGTGACATGTTC-3′ (SEQ. ID. No. 33), and forthe bottom strand primer was Fam5′-CCCCAAAATGGTCAGAGAAACCTTTATCTGTATC-3′ (SEQ. ID. No. 34).

The PCR reaction was performed at 94° C. for 2 min, followed by theaddition of AmpliTaq DNA polymerase, and, then, 30 cycles of 94° C. for15 sec, 60° C. for 2 min. A final extension was then performed at 72° C.for 7 min. A wild-type PCR fragment from genomic DNA was then mixed withthe mutant PCR fragment in a ratio of 1:1. In order to remove Taq DNApolymerase, 1 μl of proteinase K was added (20 mg/ml, QIAGEN) for every12 μl of PCR products. This reaction was incubated at 70° C. for 10 minand at 80° C. for 10 min to inactivate the Proteinase K. Heteroduplexfragments were then formed by heating the mixture at 94° C. for 1 min,65° C. for 15 min, and then cooling down to room temperature. The PCRfragments generated from the genomic DNA of cell line SW1116 did notrequire the addition of wild type PCR fragments.

For the cleavage reaction, the standard reaction mixture consisted of 10mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 5 mM MgCl₂, 5% DMSO, 1.5 Mbetaine, 100 ng PCR products, 500 nM, and purified Tma endonuclease Vprotein. The reaction mixture was incubated at 65° C. for 1 hour and wasterminated by adding 15 μl of reaction mixture to 2 μL of 10× ligasesupplemental buffer (200 mM Tris-HCl, pH 8.5, 12.5 mM MgCl₂, 500 mM KCl,100 mM DTT, and 200 μg/ml BSA). The KCl of the 10× ligase supplementalbuffer is responsible for the inhibition of Tma endoV cleavage activity.Next, 1 μl of 20 mM NAD⁺, and 2 μl of 60 nM AK16D DNA ligase was addedto bring the final concentration of the ligase reaction mixture to 20 mMTris-HCl pH 7.6, 5 mM MgCl₂, 50 mM KCl, 10 mM DTT, 1 mM NAD⁺, 20 μg/mlBSA, and 6 nM AK16D DNA ligase. The mixture was incubated at 65° C. for20 min and terminated by adding an equal volume of GeneScan stopsolution (50 mM EDTA, 1% blue dextran, and 80% formamide). The reactionmixture was then heated at 94° C. for 2 min. and cooled on ice. 3 μl ofthe mixture were loaded onto a 6% denaturing polyacrylamide gel andelectrophoresed for 1 hr on an ABI 377 sequencer (Perkin Elmer) at 1000volts, 60 mA, 200 W, and a gel temperature of 45° C.

After endoV cleavage, none of the major cleavage bands were found in thepure wild type and pure mutant G12V. Two major cleavage bands were foundin the sample of G12+G12V and G12+G12D (LS180). The length of the FAMlabeled (blue band; top strand cleavage product) is about 157-160 bpwhich is in the range of the expected length of 159 bp for a mismatchcleavage. The length of TET labeled (green band; bottom strand cleavageproduct) is around 118 bp which is also close to the expected value of116 bp for a mismatch cleavage. In addition to the G12V mutation,mutations G12D and G12A were also detected in similar assays. In theG12D assay, both top and bottom strand cleavage products were observed,and their intensities were only a little less than that of G12V. In thedetection of G12A, the cleavage of both strands was again observed. Theintensity of the top strand product was almost the same as that of G12D,but the intensity of the bottom strand product was much lower than thoseof the other two mutants. The mutation G13D was not detected.

FIG. 13 demonstrates how the addition of the ligation step dramaticallydecreases background due to non specific nicking. For this assay, PCRfragments were generated from DNA samples that were homozygous foreither the wt or mutation at position G12 in kras, or heterozygous atthis position. Without the ligation step, nonspecific cleavage isobserved in all the samples. But with the addition of the Tsp. AK16Dligase step, this background signal is dramatically reduced. Only inheterozygous samples (i.e. the last two lanes of each set) does oneobserve cleavage associated with a heteroduplex mismatch, and theseresults demonstrate that the additional ligation step does notcompromise specific mismatch cleavage. Therefore, the additionalligation step is able to reduce background due to cleavage atnon-mismatch positions but does not ligate cleavages at mismatchcleavage sites.

In addition to the major mismatch cleavage products, some non-specificcleavage bands were also observed. There is one non-specific band in allsamples which migrates above the blue (i.e. top strand) cleavageproducts present. There are also two non-specific cleavage products, oneis blue and the other is green, which migrate with low molecular weightproducts and are present in all four samples. After incubation with 1-6nM DNA ligase, the non-specific band above the blue cleavage banddecreases in intensity, indicating the non-specific nick was sealed bythe DNA ligase. The low molecular weight non-specific bands are stillpresent even when the concentration of AK16D ligase was raised to 6 nM(See FIG. 13). This suggests that these may be non-ligatable degradationproducts or, alternatively, short fragments which have denatured fromthe substrate during the 1 hour Endo V incubation at 65° C. in organicsolvents.

In order to characterize the sensitivity of this invention,base-mismatches in PCR fragments containing K-ras exon 1 mutations wereassayed under varying ratios of mutant-to-wild type DNA. With thisinvention the efficiency of cleavage for a 1:1 mutant-to-wild type DNAratio for DNA containing K-ras mutations, G12V, G12D and G12A istypically high, medium, and low, respectively. Utilizing this set ofmutations allows one to screen for sensitivity over a wide range ofcleavage efficiencies and should more accurately reflect the usefulrange of this invention. PCR fragments containing K-ras exon1 mutations,G12V, G12D, and G12A were separately mixed with wild type PCR fragmentsin mutant-to-wild type ratios of 1:1, 1:2, 1:5, 1:10, 1:20, 1:50, and1:100. These fragment mixtures were then assayed using the abovementioned Tma EndoV and DNA ligase conditions. FIG. 14 shows the amountof cleavage products for the different ratio of mutant-to-wild type DNA.The results indicate that cleavage signals can be distinguished frombackground for mutant-to-wild type ratios of up to 1:20 for all threemutations. For ratios greater than 1:20, the signal at the correctposition was still observed; however, it is difficult to conclusivelydistinguish between cleavage products and background. From this assay,it appears that the limit of sensitivity for the present invention isapproximately 1:20 (mutant-to-wild type ratio).

Example 16 Detection of Various Point Mutations in p53 and VHL Genes

To study the versatility of endoV to scan different point mutations, thetechnique of the present invention was applied to genomic DNA withvarious point mutations in p53 and VHL genes. Eleven samples containingpoint mutations in exon 5, 6, 7, and 8 in p53 and 13 samples containingpoint mutations in VHL were obtained. The sequence of PCR primers foramplifying exons in p53 and VHL genes are listed in Table 2A. PCRthermocycle conditions are listed in Table 2B. From the gel image of the500 bp VHL exon 1 fragment, a smear was observed, extending from about180 to 500 bp. This makes it very hard to observe the cleavage productsin this region due to the very high GC content in the middle of exon 1of VHL. As a result, interactions between PCR fragments and otherregions of the genomic DNA may occur. If so, the template should bediluted in the second round of PCR amplification to reduce the smear.Therefore, for the second round of PCR amplification, only 1 μL of a 100fold dilution of the PCR products was used and amplified under the samecycling conditions as discussed previously, but this time for only 15rounds. The smear was dramatically reduced, and the products were thenvisible.

Genomic DNA containing p53 mutations was extracted from tumor samplesand PCR amplified. Subsequently, wild-type PCR fragments were added sothat equal amounts of mutant and wild type PCR fragments were present.Genomic DNA containing the VHL gene mutation was from heterozygoussamples, so wild type PCR fragments were not added. In order to removeTaq DNA polymerase, 1 μl of proteinase K was added (20 mg/ml, QIAGEN)for every 12 μl of PCR products. This reaction was incubated at 70° C.for 10 min and at 80° C. for 10 min to inactivate the Proteinase K.Heteroduplexed fragments were then formed by heating the mixture at 94°C. for 1 min, 65° C. for 15 min, and then cooling down to roomtemperature.

For the cleavage reaction, the standard reaction mixture consisted of 10mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 5 mM MgCl₂, 5% DMSO, 1.5 Mbetaine, 100 ng PCR products, 500 nM, and purified Tma endonuclease Vprotein. The reaction mixture was incubated at 65° C. for 1 hour and wasterminated by adding 15 μl of reaction mixture to 2 μL of 10× ligasesupplemental buffer (200 mM Tris-HCl, pH 8.5, 12.5 mM MgCl₂, 500 mM KCl,100 mM DTT and 200 μg/ml BSA). Next, 1 μl of 20 mM NAD⁺ and 2 μl of 60nM AK16D DNA ligase were added to bring the final concentration of theligase reaction mixture to 20 mM Tris-HCl pH 7.6, 5 mM MgCl₂, 50 mM KCl,10 mM DTT, 1 mM NAD⁺, 20 μg/ml BSA, and 6 nM AK16D DNA ligase. Themixture was incubated at 65° C. for 20 min and terminated by adding anequal volume of GeneScan stop solution (50 mM EDTA, 1% blue dextran, and80% formamide). The reaction mixture was then heated at 94° C. for 2min. and cooled on ice. Three μl of the mixture were loaded onto a 6%denaturing polyacrylamide gel and electrophoresed for 1 hr on an ABI 377sequencer (Perkin Elmer) at 1000 volts, 60 mA, 200 W, and a geltemperature of 45° C.

A summary of the mutation scanning results by this method are providedin Table 4. TABLE 4 Summary of Tma EndoV/Ligase mutation scanning oncancer genes. Surrounding Cleavage Gene Exon Mutation sequences ChangeTop Strand Bottom Strand K-ras Exon 1 G12V TGGTG G→T +++ ++ G12D TGGTGG→A ++ ++ G12A TGGTG G→C ++ + G13D TGGCG G→A − − APC Exon 15 I1307KAATAA T→A + ++ p53 Exon 5 C135Y TTGCC G→A ++ − R175H GCGCT G→A − − Exon6 R196C TCCGA C→T − ++ Y220C CTATG A→G ++ − Exon 7 S241F TTCCT C→T + +G245S GCGGC G→A +++ − R248Q CCGGA G→A +++ − R248W ACCGG C→T − ++ Exon 8R273H GCGTG G→A + + R273C TGCGT C→T − − R282W ACCGG C→T ++ ++ BRCA 1Exon 2 185 del.AG TT AG AG AG deletion +++ +++ Exon 20 5382 Ins.C AT CCC C insertion + − BRCA 2 Exon 11 6174de1.T AG T GG T deletion + − VHLExon 1 P157L GCCCG C→T − ++ W159A TATGG T→A +++ ++ G164D CGGCG G→A − −Y169H CCTAC T→C − + Y183H GCTAC T→C ++ ++ Exon 2 F19OL TTCAG C→G − ++G198G GGGCT G→T ++ +++ L199F GGCTT C→T − +++ S200 Ins.E CT AGA GG AGAinsertion +++ +++ A220T TTGCC G→A +++ − N221 del.A CC A AT A deletion+++ +++ Exon 3 L229P TCTGA T→C − + R232Q GCGAT G→A + ++ R238W TCCGG C→T− ++ L259Q TCTGG T→A +++ ++*: (+++): high intensity cleavage. (++); intermediate intensitycleavage. (+) low intensity cleavage (−): no cleavage observed.

For the p53 gene, 9 out of the 11 mutations were detected. For the VHLgene, 12 out of 13 point mutations were detected. For all of the pointmutations not detected, DNA sequencing was performed to PCR fragments ofthese samples in order to verify the presence of the mutation and theratio of mutant to wild type DNA. The results showed that the mutationswere present in these samples and the ratio of mutant to wild type DNAwas approximately 1:1 in all of the samples, except for sample R175H.After adding wild-type PCR fragments to the mutant PCR fragmentscontaining R175H, cleavage assays were repeated, but the results forR175H were still negative.

All but four mutations listed in Table 4 could be recognized by Tmaendonuclease V. The four non-detectable mutations are K-ras G13D(G→A),p53 R175H(G→A), R273C(C→T), and VHL G164D(G→A) (Table 4), and thesurrounding sequence of each mutation for both top and bottom strand arelisted in Table 5. TABLE 5 List of Mutations and surrounding sequencesfor top and bottom strand, with cleavage intensity. SurroundingSurrounding Sequences Sequences Gene Exon Mutation (Wt) (Mt) ChangesMismatches Purine (G or A) containing strand VHL Exon 2 L199F AAGCCAAACC G→A G:T, A:C +++ VHL Exon 3 R232Q GCGAT GCAAT G→A G:T, A:C + P53Exon 5 R175H GCGCT GCACT G→A G:T, A:C − P53 Exon 8 R273C ACGCA ACACA G→AG:T, A:C − P53 Exon 7 G245S GCGGC GCAGC G→A G:T, A:C +++ P53 Exon 7R248Q CCGGA CCAGA G→A G:T, A:C +++ VHL Exon 3 R238W CCGGA CCAGA G→A G:T,A:C ++ P53 Exon 7 R248W CCGGT CCAGT G→A G:T, A:C ++ P53 Exon 8 R282WCCGGT CCAGT G→A G:T, A:C ++ VHL Exon 3 L229P TCAGA TCGGA A→G A:C, G:T +P53 Exon 6 R196C TCGGA TCAGA G→A G:T, A:C ++ P53 Exon 8 R273H GCGTGGCATG G→A G:T, A:C + P53 Exon 7 S241F AGGAA AGAAA G→A G:T, A:C + VHLExon 1 G164D CGGCG CGACG G→A G:T, A:C − K-ras Exon 1 G13D TGGCG TGACGG→A G:T, A:C − VHL Exon 1 P157L CGGGC CGAGC G→A G:T, A:C ++ K-ras Exon 1G12D TGGTG TGATG G→A G:T, A:C ++ VHL Exon 2 A220T TTGCC TTACC G→A G:T,A:C +++ P53 Exon 5 C135Y TTGCC TTACC G→A G:T, A:C ++ VHL Exon 1 Y169HGTAGG GTGGG A→G A:C, G:T + VHL Exon 1 Y183H GTAGC GTGGC A→G A:C, G:T ++P53 Exon 6 Y220C CTATG CTGTG A→G A:C, G:T ++ VHL Exon 2 F190L CTGAACTCAA G→C G:G, C:C ++ K-ras Exon 1 G12A TGGTG TGCTG G→C G:G, C:C ++K-ras Exon 1 G12V TGGTG TGTTG G→T G:A, T:C +++ VHL Exon 2 G198G GGGCTGGTCT G→T G:A, T:C ++ VHL Exon 1 W159A CCATA CCTTA A→T A:A, T:T ++ VHLExon 3 L259Q CCAGA CCTGA A→T A:A, T:T ++ APC Exon 15 I1307K TTATT TTTTTA→T A:A, T:T ++ P53 Exon 6 Y220C CATAG CACAG T→C T:G, C:A − K-ras Exon 1G12D CACCA CATCA C→T C:A, T:G ++ P53 Exon 8 R273H CACGC CATGC C→T C:A,T:G + VHL Exon 1 Y169H CCTAC CCCAC T→C T:G, C:A − VHL Exon 1 Y183H GCTACGCCAC T→C T:G, C:A ++ VHL Exon 1 P157L GCCCG GCTCG C→T C:A, T:G − P53Exon 7 G245S GCCGC GCTGC C→T C:A, T:G − VHL Exon 3 L229P TCTGA TCCGA T→CT:G, C:A − P53 Exon 6 R196C TCCGA TCTGA C→T C:A, T:G − P53 Exon 7 R248QTCCGG TCTGG C→T C:A, T:G − VHL Exon 3 R238W TCCGG TCTGG C→T C:A, T:G −P53 Exon 7 R248W ACCGG ACTGG C→T C:A, T:G − P53 Exon 8 R282W ACCGG ACTGGC→T C:A, T:G ++ VHL Exon 2 A220T GGCAA GGTAA C→T C:A, T:G − P53 Exon 5C135Y GGCAA GGTAA C→T C:A, T:G − K-ras Exon 1 G13D CGCCA CGCCA C→T C:A,T:G − VHL Exon 1 G164D CGCCG CGTCG C→T C:A, T:G − VHL Exon 2 L199F GGCTTGGTTT C→T C:A, T:G − P53 Exon 5 R175H AGCGC AGTGC C→T C:A, T:G − P53Exon 8 R273C TGCGT TGTGT C→T C:A, T:G − P53 Exon 7 S241F TTCCT TTTCT C→TC:A, T:G + VHL Exon 3 R232Q ATCGC ATTGC C→T C:A, T:G ++ VHL Exon 2 F190LTTCAG TTGAG C→G C:C, G:G − K-ras Exon 1 G12A CACCA CAGCA C→G C:C, G:G +K-ras Exon 1 G12V CACCA CAACA C→A C:T, A:G ++ VHL Exon 2 G198G AGCCCAGACC C→A C:T, A:G +++ VHL Exon 1 W159A TATGG TAAGG T→A T:T, A:A +++ VHLExon 3 L259Q TCTGG TCAGG T→A T:T, A:A +++ APC Exon 15 11307K AATAA AAAAAT→A T:T, A:A + VHL Exon 2 F190L CTGAA CTCAA G→C G:G, C:C ++ VHL Exon 2F190L TTGAG TTCAG G→C G:G, C:C − K-ras Exon 1 G12A TGGTG TGCTG G→C G:G,C:C ++ K-ras Exon 1 G12A CAGCA CACCA G→C G:G, C:C + K-ras Exon 1 G12VTGGTG TGTTG G→T G:A, T:C +++ VHL Exon 2 G198G GGGCT GGTCT G→T G:A, T:C++ VHL Exon 2 G198G AGACC AGCCC A→C A:G, C:T +++ K-ras Exon 1 G12V CAACACACCA A→C A:G, C:T ++ APC Exon 15 I1307K AAAAA AATAA A→T A:A, T:T + VHLExon 1 W159A TAAGG TATGG A→T A:A, T:T +++ VHL Exon 3 L259Q TCAGG TCTGGA→T A:A, T:T +++ VHL Exon 3 L259Q CCAGA CCTGA A→T A:A, T:T ++ VHL Exon 1W159A CCATA CCTTA A→T A:A, T:T ++ APC Exon 15 I1307K TTATT TTTTT A→ A:A,T:T ++

Previous assays with double-stranded oligonucleotide substratesdemonstrated that every point mutations can be detected with this method(FIGS. 3 and 5, Table 4), with mismatches containing cytosine to be theleast efficiently cleaved. Even though all four of these mutationsgenerate heteroduplex PCR fragments with cytosine mismatches, so do thevast majority of the mutations surveyed. Therefore, the presence of acytosine mismatch is not sufficient to explain why these four mutationswere not detected. Instead, the data suggests that flanking sequencesneed to be taken into account. Analysis of the surrounding sequences ofthe four mutations which could not be cleaved by Tma EndoV gives thefollowing consensus sequences: gRcg, rcRc, cgYc, and gYgy. The positionof the mutation or polymorphism is underlined and shown in upper case,the last two sequences listed are the complements of the first twosequences (where R and Y stand for a purine and pyrimidine base,respectively). In a very preliminary analysis, the 1^(st) 100 randomsingle nucleotide polymorphisms (SNPs) from human chromosome 22 weresearched for the four refractory sequences, and only one, rcRc, wasfound (Table 6). TABLE 6 The frequency of Tma EndoV refractory sequencesin Human Chromosome 22, 1^(st) 100 random SNPs. EndoV Refractory SitesCG dinucleotides Transitions Transversions gRcg rcRc cgYc gYgy cR Yg R YW, S M, K acRca gcRga aaYgg agRgg ttYat caSaa ggMcc acRaa acYgt agRtgtcYtc aaWta ccMaa ccRgc agYga tgRaa gcYtc aaWaa caKcg gcRat ggYgg ctRattaYct aaSta gtKcc ccRtg aaYgc agRga caYaa taStt acMat tcRaa agYga agRggatYtt taSct atMaa tcRtg tcYgg taRtc tgYag tgWga tgKgt acRtg gcYtc gtRtgaaYat ttStt aaMtt caYgt aaRtt caYtg aaSgt ttMac acYgt agRgc taYca caSaggaKgg aaYgc aaRag taYaa caSat acMgt gtYgg gaRgc caYtg acSgt taMcg ttYgtggRgg tcYac tcScg ttKcc ccYgc atRca ttYag agWta ggMac caYgt gcYtc gtWcactKta ctYgg ccYat aaWct agMgg ggYga ctYtg ccSgt agMga caYgt aaYtt ttWgaagMgg ccYca ccSgt tgYtg gtStg acWtg Total is: 0 1 0 0 8 18 14 20 21 18Subsequent computer searches of some 4,000 SNPs in the SNP databaserevealed the frequency of the gRcg (and complement cgYc) was about 0.1%and the rcRc (and complement gYgy) was about 2%. This preliminary resultsuggests that the frequency of the four refractory sequences is very low(2%), and it is possible that Tma endoV can cleave approximately 98% ofthe poymorphisms and mutations found in the human genome.

In addition to detecting single base mutations, the analysisdemonstrates that the present invention is also capable of detectingsmall insertions and deletions (See Table 4). A three base insertion inthe VHL gene and a two base deletion in BRCA 1 both resulted in verystrong cleavage signals for both strands. A single base deletion in theVHL gene also resulted in a strong signal for both strands. The worstresults were obtained with a single insertion and a single base deletionin the BRCA 1 gene. Both for the insertion and deletion only one strandwas observed to be cleaved, and the signal was relatively weak for bothcases. Despite this lower efficiency, this type of signal is capable ofconfirming a variation in an unknown sample. These results demonstratethat the present invention can detect small insertions and deletions,and, similar to mutation detection, the efficiency is sequencedependent.

Example 17 Detection of TΠA Mutation in Codon 1307 of Exon 15 in the APCGene

The method of the present invention was applied to clinical samples byscanning T→A mutation in codon 1307 in exon 15 of APC gene. Nine genomicsamples from colon cancer tissues and four wild type samples wereassayed.

The sequence of the top strand PCR primer was (SEQ. ID. No. 35)Tet-5′-CCCCGCTGCCACTTGCAAAGTTTCTTC-3′

while the sequence of the bottom strand PCR primers was (SEQ. ID. No.36) Fan-5′-CCCCACTCTGAACGGAGCTGGCAAT-3′

The PCR reaction condition was listed in Table 2B. Since genomic DNAcontaining APC mutations were heterozygous, it is not necessary to addwild-type PCR fragments. In order to remove Taq DNA polymerase, 1 μl ofproteinase K was added (20 mg/ml, QIAGEN) for every 12 μl of PCRproducts. This reaction was incubated at 70° C. for 10 min and at 80° C.for 10 min to inactivate the Proteinase K. Heteroduplex fragments werethen formed by heating the mixture at 94° C. for 1 min, 65° C. for 15min, and then cooling down to room temperature. The PCR fragments fromgenomic DNA of cell line SW1116 were directly heated and cooled down toproduce heteroduplexes without adding wild type PCR fragments.

For the cleavage reaction, the standard reaction mixture consisted of 10mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 5 mM MgCl₂, 5% DMSO, 1.0 Mbetaine, 100 ng PCR products, 500 nM, and purified Tma endonuclease Vprotein. The reaction mixture was incubated at 65° C. for 1 hour and wasterminated by adding 15 μl of reaction mixture to 2 μL of 10× ligasesupplemental buffer (200 mM Tris-HCl, pH 8.5, 12.5 mM MgCl₂, 500 mM KCl,100 mM DTT, and 200 μg/ml BSA). Next, 1 μl of 20 mM NAD⁺ and 2 μl of 60nM AK16D DNA ligase was added to bring the final concentration of theligase reaction mixture to 20 mM Tris-HCl pH 7.6, 5 mM MgCl₂, 50 mM KCl,10 mM DTT, 1 mM NAD⁺, 20 μg/ml BSA, and 6 nM AK16D DNA ligase. Themixture was incubated at 65° C. for 20 min and terminated by adding anequal volume of GeneScan stop solution (50 mM EDTA, 1% blue dextran, and80% formamide). The reaction mixture was then heated at 94° C. for 2min. and cooled on ice. Three μl of the mixture were loaded onto a 6%denaturing polyacrylamide gel and electrophoresed for 1 hr on an ABI 377sequencer (Perkin Elmer) at 1000 volts, 60 mA, 200 W, and a geltemperature of 45° C. The correct mutations was detected in all of thesamples no false positives were detected in the wild-type or tumorsamples. The cleavage activity was higher in the presence of 1.0 Mbetaine than that in 1.5 M betaine (FIG. 10B), presumably due to the ATrich nature of exon 15 of the APC gene. Therefore, the amount of betainemay need to be adjusted, and the GC-content of the region amplified mayact as a guide.

Example 18 Detection of k-ras Mutations in the Diluted Samples

In order to determine the sensitivity of this invention, PCR fragmentscontaining K-ras exon 1 mutations, G12V, G12D, and G12A were used astemplates, and the mutation detection abilities of the present inventionwere assayed in different ratios of mutant-to-wild type DNA ranging from1:1 to 1:100. The initial ratio in the G12A assay was 0.7:1, because themutant-to-wild type ratio in the genomic DNA containing the G12Amutation was 0.7:1 and the pure mutant G12A was not available. Thesequences of PCR primers are listed in Table 2A, and the PCR thermocycleconditions are listed in Table 2B. In order to remove Taq DNApolymerase, 1 μl of proteinase K was added (20 mg/ml, QIAGEN) for every12 μl of PCR products. This reaction was incubated at 70° C. for 10 minand at 80° C. for 10 min to inactivate the Proteinase K. Heteroduplexfragments were then formed by heating the mixture at 94° C. for 1 min,65° C. for 15 min, and then cooling down to room temperature.

The PCR fragments containing K-ras exon1 mutations, G12V, G12D, and G12Awere individually mixed with wild type PCR fragments in the ratio ofmutant-to-wildtype of 1:1, 1:2, 1:5, 1:10, 1:20, 1:50, and 1:100. Forthis assay, the total amount of PCR fragments was held constant.Therefore, 100 ng of heteroduplex PCR fragments with differentmutant-to-wild type ratios were cleaved with Tma endoV and ligated withTsp. AK16D DNA ligase.

For the cleavage reaction, the standard reaction mixture consisted of 10mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 5 mM MgCl₂, 5% DMSO, 1.5 Mbetaine, 100 ng PCR products, 500 nM, and purified Tma endonuclease Vprotein. The reaction mixture was incubated at 65° C. for 1 hour and wasterminated by adding 15 μl of reaction mixture to 2 μL of 10× ligasesupplemental buffer (200 mM Tris-HCl, pH 8.5, 12.5 mM MgCl₂, 500 mM KCl,100 mM DTT, and 200 μg/ml BSA). Next, 20 mM NAD⁺ and 2 μl of 60 nM AK16DDNA ligase were added to bring the final concentration of the ligasereaction mixture to 20 mM Tris-HCl pH 7.6, 5 mM MgCl₂, 50 mM KCl, 10 mMDTT, 1 mM NAD⁺, 20 μg/ml BSA, and 6 nM AK16D DNA ligase. The mixture wasincubated at 65° C. for 20 min and terminated by adding an equal volumeof GeneScan stop solution (50 mM EDTA, 1% blue dextran, and 80%formamide). The reaction mixture was then heated at 94° C. for 2 min.and cooled on ice. Three μl of the mixture were loaded onto a 6%denaturing polyacrylamide gel and electrophoresed for 1 hr on an ABI 377sequencer (Perkin Elmer) at 1000 volts, 60 mA, 200 W, and a geltemperature of 45° C. Since a TAMRA labeled GeneScan Molecular sizestandard 500 was loaded on the same gel, the molecular weight ofcleavage products could be estimated by comparison to the mobility ofsize standard using the GeneScan analysis software versions 2.1 or 3.0a.

FIG. 14 shows the amount of cleavage products for the different ratiosof mutant-to-wild type DNA. The peak area was measured by the GeneScansoftware (v. 3.0) and was used for determining the relative fluorescenceintensity. Cleavage signals generated wild type homoduplex were used aswild type controls. Background intensity was determined by measuringsignal intensities directly above and below the cleavage band for bothtop and bottom strand products, respectively. The background signalswere then averaged from seven samples with different ratios and one wildtype sample. The peak area associated with the cleavage product wasdetermined by analysis with GeneScan Analysis software 3.0. Bar graphsin FIG. 14 indicate the relative fluorescence intensity with theirrespective mutant-to-wild type ratios. (Striped bar: top strand cleavageproducts; solid bar: bottom strand cleavage products.) The averagebackground signals are indicated with a horizontal dashed line.(Bkdg-top stands for background signals for the average top strandcleavage products, bkdg-bottom stands for the average background signalsfor bottom strand cleavage products.) The mutation, nucleotide change,and the mismatch base pairs are indicated inside the graph.

These results indicate that the present invention is able toconsistently distinguish cleavage signals from background signals inmutant to wild-type DNA ratios of at least 1:20 for all three mutants.The present invention has been able to detect cleavage signals in mutantto wild-type DNA ratios as high as 1:50, but this has not been observedin all cases studied. Therefore, the present invention is limited to asensitivity of 1:20 (mutant-to-wild type DNA).

Example 19 Detection of Small Deletion, Insertion in BRCA1, BRCA2, andVHL Genes

In order to determine the ability of the present invention to detectsmall deletion or insertions, it was applied to samples containing an AGdeletion in exon 2 or a C insertion in exon 20 of BRCA 1, a T deletionin exon 11 of the BRCA 2 gene, or either an AGA insertion or an Adeletion in exon 2 of von Hippel Lindau (VHL) gene.

The sequence of PCR primers for amplifying exon 2 and 20 in BRCA1, exon11 in BRCA2 and exon 2 in VHL are listed in Table 2A, and PCRthermocycle conditions are listed in Table 2B. Since the mutations areheterozygous in the sample, it was not necessary to add wild-type PCRfragments. In order to remove Taq DNA polymerase, 1 μl of proteinase Kwas added (20 mg/ml, QIAGEN) for every 12 μl of PCR products. Thisreaction was incubated at 70° C. for 10 min and at 80° C. for 10 min toinactivate the Proteinase K. Heteroduplex fragments were then formed byheating the mixture at 94° C. for 1 min, 65° C. for 15 min, and thencooling down to room temperature.

For the cleavage reaction, the standard reaction mixture consisted of 10mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 5 mM MgCl₂, 5% DMSO, 1.5 Mbetaine, 100 ng PCR products, and 500 nM purified Tma endonuclease Vprotein. The reaction mixture was incubated at 65° C. for 1 hour and wasterminated by adding 15 μl of reaction mixture to 2 μL of 10× ligasesupplemental buffer (200 mM Tris-HCl, pH 8.5, 12.5 mM MgCl₂, 500 mM KCl,100 mM DTT, and 200 μg/ml BSA). Next, 1 μl of 20 mM NAD⁺ and 2 μl of 60nM AK16D DNA ligase were added to bring the final concentration of theligase reaction mixture to 20 mM Tris-HCl pH 7.6, 5 mM MgCl₂, 50 mM KCl,10 mM DTT, 1 mM NAD⁺, 20 μg/ml BSA and 6 nM AK16D DNA ligase. Themixture was incubated at 65° C. for 20 min and terminated by adding anequal volume of GeneScan stop solution (50 mM EDTA, 1% blue dextran and80% formamide). The reaction mixture was then heated at 94° C. for 2min. and cooled on ice. 3 μl of the mixture were loaded onto a 6%denaturing polyacrylamide gel and electrophoresed for 1 hr on an ABI 377sequencer (Perkin Elmer) at 1000 volts, 60 mA, 200 W, and a geltemperature of 45° C. Because a TAMRA labeled GeneScan Molecular sizestandard 500 was loaded on the same gel, the molecular weight ofcleavage products could be estimated by comparison to the mobility ofsize standard using the GeneScan analysis software versions 2.1 or 3.0a.

The gel image showed that the cleavage of the AG deletion mismatch ofBRCA 1 was very strong, but the cleavage bands associated with the Cinsertion of BRCA 1 and the T deletion of BRCA 2 are faint (FIG. 15). Anon-specific band below the cleavage band of the T deletion was removedafter ligation, which indicates that non-specific nicks can be sealed bythe ligase. For the VHL gene, the cleavage of the AGA insertion is verystrong, and the cleavage of an A insertion is also good (Table 4). Theseresults demonstrate that endoV can recognize and cleave a two to threenucleotide insertion or deletion efficiently. It also can detect a onenucleotide insertion or deletion, but the efficiency was observed to beless than that of a two or three nucleotide change.

A comparison of lanes 13-18 with lanes 19-24 demonstrate the advantagesof using a DNA ligase step for distinguishing point mutations in the p53gene when using clinical samples. Significant background signal in lanes13-18 is all but eliminated in lanes 19-24 allowing for unambiguousdetection of mutant signals.

Example 20 Detection of Mutations in Long PCR Fragments

The ability of the present invention to mutation scan on longer PCRfragments was determined using a 1.7 kb PCR fragment from the p53 gene.The sequences of the PCR primers used to amplify this 1.7 kb segment arelisted in Table 2A, while PCR thermocycle conditions are listed in Table2B. In order to remove Taq DNA polymerase, 1 μl of proteinase K wasadded (20 mg/ml, QIAGEN) for every 12 μl of PCR products. This reactionwas incubated at 70° C. for 10 min and at 80° C. for 10 min toinactivate the Proteinase K. Heteroduplex fragments were then formed byheating the mixture at 94° C. for 1 min, 65° C. for 15 min, and thencooling down to room temperature. The PCR fragments were then washedtwice with 10 mM Tris pH 7.5 and excess dNTP and primers were removed bya Microcon 30 filter (Millipore) centrifugation step before endoVcleavage reaction. This resulted in the buffer condition changing andthe volume being reduced to half of the original volume before washing.Therefore, the DNA concentration was increased two fold to about 10-20ng/μl. The Tma endoV cleavage reaction was performed at 65° C. for 1hour in the buffer containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2%glycerol, 200 ng PCR heteroduplex fragments, 5 mM MgCl₂, 1.5 M betaine,2% DMSO, and 500 nM Tma endoV. After the endoV reaction, the fragmentswere washed again to remove betaine and DMSO. The ligation was performedat 65° C. for 20 min in the presence of 1-6 nM Tsp. AK16D ligase in abuffer containing 20 mM Tris-HCl (pH7.6), 5 mM MgCl₂, 50 mM KCl, 10 mMDTT, 1 mM NAD⁺, and 20 μg/ml BSA.

This procedure was performed using genomic DNA containing p53 R248W(C→T) as the template, and 400 bp, 800 bp, 1.2 kb and 1.7 kb fragmentscontaining the R248W mutation were amplified. This assay demonstratesthat the invention can detect mutations in PCR fragments up to 1.7 kb insize and potentially higher (FIG. 16). Top strand cleavage (TET labeled)was also observed in these assays.

Example 21 Micro DNA Sequencing to Detection of P53 R248w(CΠT)

The strategy of micro-DNA sequencing is illustrated in FIGS. 16A and18A. The DNA fragment containing p53 R248W(C→T) was PCR amplified withunlabeled primers. The sequences of PCR primers are listed in Table 2A,and the PCR thermocycle conditions are listed in Table 2B. PCR fragmentscontaining p53 R248W(C→T) were mixed with equal amounts of wild type PCRfragments. In order to remove Taq DNA polymerase, 1 μl of proteinase Kwas added (20 mg/ml, QIAGEN) for every 12 μl of PCR products. Thisreaction was incubated at 70° C. for 10 min and at 80° C. for 10 min toinactivate the Proteinase K. Heteroduplex fragments were then formed byheating the mixture at 94° C. for 1 min, 65° C. for 15 min, and thencooling down to room temperature. The PCR fragments were then washedtwice with 10 mM Tris pH 7.5 and excess dNTP and primers were removed bya Microcon 30 filter (Millipore) centrifugation step before endoVcleavage reaction.

The Tma endoV cleavage reaction was performed at 65° C. for 1 hour inthe buffer containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol, 200ng PCR heteroduplex fragments, 5 mM MgCl₂, 1.5 M Betaine, 2% DMSO, and500 nM Tma endoV. After the endoV reaction, the fragments were washedagain to remove betaine and DMSO. The ligation was performed at 65° C.for 20 min in the presence of 1-6 nM Tsp. AK16D ligase in a buffercontaining 20 mM Tris-HCl (pH7.6), 5 mM MgCl₂, 50 mM KCl, 10 mM DTT, 1mM NAD⁺, and 20 μ/ml BSA. After ligation, the mixture was incubated at65° C. for 20 min and then washed to remove salts.

The nicked heterduplex products were excised several nucleotides backfrom the nick 3′→5′ with DNA polymerase I Klenow fragment. The reactionmixture (40 μl) contained 120 ng DNA from the ligation reaction, 1× E.coli.DNA polymerase I buffer, and 20 units DNA polymerase I Klenowfragment. The mixture was incubated at 37° C. for 30 min. The product ofthis reaction was subsequently used as substrate for a sequencingreaction.

The DNA sequencing reaction contained 30 ng DNA from the Klenow fragmentreaction, 20 μM dNTP, 20 μM ddGTP, 224 μM ddTTP, 22 μM ddATP, 32 μMddCTP, 1× sequencing buffer, and 1 μl of Taq DNA polymerase FS (PerkinElmer lot # 361451005). The ratio of the four dideoxynucleotide(A:C:G:T=0.11:0.16:0.10:1.12) was taken from Rosenblum, B. B. et al.,“New Dye-Labeled Terminators for Improved DNA Sequencing Pattern,”Nucleic Acids Res. 25:4500-4 (1997), which is hereby incorporated byreference. The reaction mixture was incubated at 60° C. for 30 sec usingPE GeneAmp PCR system 2400 thermocycler. The excess BigDyedideoxynucleotide was removed by loading the reaction mixture on aCentri-Sep™ spin column P/N CS-90 (Princeton Separation, Adelphia, N.J.)and microcentrifuged at 3000 rpm for 2 min. Five μl of the eluate wasadded to an equal volume of GeneScan stop solution (50 mM EDTA, 80%formamide, and 1% blue dextran). After heating at 94° C. for 2 min, 1 μlof the mixture was loaded on a 10% denaturing polyacrylamide gel andelectrophoresed at 1000 volts, 60 mA, 150 W, and a gel temperature of45° C. Running time took approximately 4 hours. The Run Module was setto filter E and the Matrix was set to dRhodamine Matrix. The data wasanalyzed with GeneScan analysis (v.3.0a). See FIG. 16B.

For comparison of the sequence results, standard dRhodamine DNA cyclingsequencing was also performed. The DNA template for this sequencingreaction was PCR product which was amplified with unlabeled PCR primers.The sample was then washed twice with 10 mM Tris pH 7.5 and excessprimers were removed by a Microcon 30 filter (Millipore) centrifugationstep. One of the unlabelled PCR primers was then added to be used as asequencing primer. Standard DNA sequencing was performed using an ABIPrism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer). Twenty μl of the reaction mixture, containing 8.0 μl ofterminator ready reaction mix, 6.0 μl of PCR products (10 ng/l), 1.0 μlof each PCR primer (3.2 μM), and 7 μl of ddH₂O, was incubated in a PEGene Amp PCR System 2400 thermocycler for 25 cycles of 96° C. for 10sec, 50° C. for 5 sec, and 60° C. for 4 min. After the reaction, themixture was precipitated by adding 2 μl of 3 M NaAc (pH 4.6) and 50 μlof 95% ethanol, it was placed on ice for 10 min. After microcentrifugingfor 15-20 min at maximum speed, the pellets was rinsed with 70% ethanoland dried. The pellet was then suspended in 4 μl of DNA sequencingloading buffer (5 mM EDTA, 1% blue dextran, and 80% formamide). Afterheating at 95° C. for 2 min., 1-2 μl of the mixture was loaded on a 10%polyacrylamide denaturing gel (small plate) and electrophoresed at 1000volts, 60 mA, 150 W, and a gel temperature of 45° C. Running time wasapproximately 4 hours on an ABI DNA sequencer 377. The Run Module wasset to filter E, and Dye Set/Primer File was set to DT {dR SetAny-primer}. The sequencing patterns were analyzed with DNA sequencinganalysis software (v. 3.0)(see FIG. 18).

The sequence results for the bottom strand are shown in FIG. 18 B(micro-DNA sequencing) and FIG. 18C (standard DNA sequencing). Theposition where G (blue color) and A (green color) peaks overlapindicates the position of the mismatch, and shows that the mutation isG→A (C→T for the top strand).

Example 22 Detection of K-ras G12V Mutation in PCR Fragments Containing7-deaza-dGTP

In order to explore the possibility of using nucleotide analogues withTma endonuclease v, the same concentration of 7-deaza-dgtp was usedinstead of regular dgtp in PCR reaction mixture. The PCR reaction wassetup to amplify k-ras exon-1 using genomic DNA from a cell linecontaining wild type DNA (ht29) and mutant DNA g12v(g→t) (sw620). ThePCR primers and PCR program, Tma cleavage and Tsp.DNA ligationconditions were the same as the standard conditions. To compare thecleavage products from the PCR fragment containing 7-deaza-dgtp, thecleavage products from PCR fragments containing regular dgtp were alsoloaded on the same gel.

The 7 position in 7-deaza-dGTP is a carbon instead of nitrogen (FIG.17A). The PCR reaction was setup to amplify K-ras exon-1 using genomicDNA from cell line containing wild type DNA (HT29) and mutant DNAG12V(G→T) (SW620). In comparison to the cleavage products from the PCRfragment containing 7-deaza-dGTP, the cleavage products from PCRfragments containing regular dGTP were also loaded on the same gel. Forthe heteroduplex PCR fragment containing 7-deaza-dGTP, cleavage of thetop strand containing (G→T) mutation was inhibited, while cleavage ofthe bottom strand containing (C→A) is still present (FIG. 17B). Thisresult indicated a possible contact between Tma endonuclease V and N-7in Guanine. While the use of 7-deazaG did not allow for cleavage of therefractory G13D mutation, the concept of lowering the stability of G-Cbase pairs by using a non-cleavable analogue was shown to be robust.Several additional (non-specific) cleavage products are observed in thesecond lane containing deazaG. Alternative analogues may be considered,especially those which base pair with guanosine but destabilizehybridization, such as 2-pyrimidinone instead of cytosine.

Example 23 Mutant Endonucleases

Wild type Thermotoga maritima Endo V can identify approximately 98% ofthe SNPs found in the human genome. To identify the remaining SNPs, orto achieve a more robust signal on existing SNPs or mutations, modifiedor novel EndoV enzymes may be required. This may be achieved by eitherintroducing mutation(s) into Thermotoga maritima Endo V to alter itsmismatch base specificity or by utilizing an Endo V from a differentorganism that has a different mismatch base specificity. A mutantThermotoga maritima EndoV or an alternate Endo V could be used in thesame reaction with wild-type Thermotoga maritima Endo V, to expand therepetoire of sequences which can be recognized in this assay.

Primary sequence alignment of different eukaryotic, prokaryotic, andarchea EndoV orthologs revealed a few residues which appeared to beevolutionarily conserved. Nine different site-specific mutants (D43A,Y80A, R88A, E89A, D105A, D110A, H116A, H125A and, K139A) wereconstructed, and 5 of these (Y80A, R88A, E89A, H116A, and K139A) appearto have useful altered specificity (See Table 7). TABLE 7 Summary ofenzymatic and binding activity of Tma EndoV mutants. Tma endoV Mutantswt D43A Y80A R88A E89A D105A D110A H116A H125A K139A Cleavage I/A MgCl₂++/++ −/− +++/− +++/− w/− ++/++ −/− +++/− ++/++ ++/− MnCl₂ +/+ −/− ++/−++/w +/− +/+ −/− ++/w +/+ ++/− Binding I/A MgCl₂ ++ ++ − ++ − ++ ++ +++++ +++ CaCl₂ ++ ++ − 1/2+ + ++ ++ − ++ ++ EDTA 1/2+ ++ − − + 1/2+ ++ −− − Predictions wt Catalytic Stacking O2, O6 N1 wt Catalytic O2, 6, N7Almost O2, 6, N7 Mg H-bond H-bond Mg H-bond wt H-bond binding Accept.?Doner? binding Accept.? Accept.?In addition, this analysis identified two aspartates, D43 and D105, asbeing the catalytic residues for this enzyme.

The approach used to develop useful Thermotoga maritima Endo V mutantsis based on creating site-directed mutants at positions that areinvolved in protein-substrate interactions. Thermotoga maritima Endo Vdemonstrates significantly greater cleavage activity with inosine oruracil within either a matched or mismatched substrate, as compared withmismatches containing the naturally occurring deoxyribonucleotide bases(dA, dC, dG, and dT). Therefore, one can increase the relativespecificity of the enzyme for a natural base by decreasing thespecificity of the enzyme for inosine or uracil, and/or increasing thespecificity for a natural base.

Protein specificity for a substrate typically involves interactionsbetween amino acid side chains of the enzyme and the functional groupsof the substrate. Interactions involving amino acid side chains andsubstrate moieties of different chemical composition (e.g.hydrophobic/polar interactions) greatly destabilize substrate bindingand can dramatically decrease the ability of an enzyme to utilize aspecific substrate. Since the enzyme functions in vitro withdramatically higher activity with inosine and uracil containingsubstrates and the natural occurring deoxyribonucleotide bases are lessfavorably cleaved, this strongly suggests one or more destabilizinginteractions associated with a natural base. Therefore, by mutagenizingthe enzyme at positions of enzyme-mismatch base interactions, thespecificity of the enzyme can be altered to favor naturally occurringdeoxyribonucleortide bases. A similar approach has been successful withhuman UDG, which recognizes uracil within DNA substrates. Kavli et al.,The EMBO Journal 15:3442-47 (1996), which is hereby incorporated byreference.

Residues that are involved directly in protein-substrate interactionshave a strong tendency to be conserved among enzymes of the same family.Therefore, in a primary amino acid sequence alignment, highly conservedresidues represent good candidates for mutagenesis. In order to identifypositions in Thermotoga maritima Endo V suitable for mutagenesis, theClustalW alignment algorithm with a PAM250 Residue Weight Table(Pairwise Alignment Parameters: Ktuple=1, Gap Penalty=3, Window=5, andDiagonals Saved=5; Multiple Alignment Parameters Gap Penalty=10 and GapLength Penalty=10) was used to perform a primary amino acid sequencealignment among 13 identified and putative Endo V enzymes fromthermophilic and mesophilic archeabacteria and eubacteria (i.e.Thermotoga maritima (SEQ. ID. No. 37), Pyrobaculum aerophilum (SEQ. ID.No. 38), Pyrococcus horikoshii (SEQ. ID. No 39), Pyrococcus abyssi (SEQ.ID. No. 40), Pyrococcus furiosus (SEQ. ID. No. 41), Archaeoglobusfulgidus (SEQ. ID. No. 42), Aeropyrum pernix (SEQ. ID. No. 43),Clostridium acetobutylicum (SEQ. ID. No. 44), Yersinia pestis (SEQ. ID.No. 45), Escherichia coli (SEQ. ID. No. 46), Bacillus subtilis (SEQ. ID.No. 47), Salmonella typhimurium (SEQ. ID. No. 48), and Streptomycescoelicolor (SEQ. ID. No. 49), majority sequence, top line (SEQ. ID. No.50) (FIG. 19)). Since the majority of enzymes utilized in the alignmentare putative Endo V enzymes, the mismatch specificity of most of theseenzymes is unknown. As a result, when utilizing the alignment toidentify candidate residues in Thermotoga maritima Endo V, one caneither assume that the homologous enzymes have similar or differentspecificities. If one assumes similar specificities, then residues thatare highly conserved for the majority of Endo V provides candidates.Whereas if one assumes different specificities, then positions wherethere exists two sets of highly conserved residues represent candidates.

In analysis assuming similar mismatch specificity among the family ofendo Vs, 12 residues (D43, F46, Y80, R88, E89, D105, D110, H116, R118,H125, K139, and F180) in Thermotoga maritima Endo V have been identifiedthat were 1) highly conserved for the majority of Endo V enzymes andwere 2) best suited for mutagenesis. Although aliphatic hydrocarbonchains (i.e. L, I, V, and M residues) can play a role inprotein-substrate interactions, positions of high conservation withthese residues are not listed, since they can also play a role instabilizing the hydrophobic core of the enzyme. In addition, highlyconserved prolines and glycines should be avoided for mutagenesis due tothe unique chemical and structural influences associated with theseamino acid residues. From the initial set of 12, 9 differentsite-specific mutants (D43A, Y80A, R88A, E89A, D105A, D110A, H116A,H125A and, K139A) and 5 of these mutant endo Vs (Y80A, R88A, E89A,H116A, and K139A) appear to have useful altered specificity. Theseresults demonstrate that the present invention is useful for identifyingThermotoga maritima EndoV positions to mutate in order to confer alteredspecificity. Since the chemical nature of the polar group (e.g.,hydrogen bond donator vs. hydrogen bond acceptor) can also have a stronginfluence on substrate specificity, further mutations at these positionscan facilitate the generation of a mutant Endo V enzymes that extend thecapabilities of the current invention (Table 8). TABLE 8 EndoV Mutants,Group 1. Add Remove Destabilizing Stabilizing Residue Residue ModifyStability F46 F46A F46Y F46(L, I, V, M) Y80 Y80A Y80F Y80(L, I, V, M)R88 R88A R88(L, I, V, M) R88K R88(N, Q) R88(D, E) R88(T, S) E89 E89AE89(L, I, V, M) E89D E89(T, S) E89(R, K) E89(Q, N) H116 H116A H116(L, I,V, M) H116(R, K) H116(N, Q) H116(D, E) H116(T, S) R118 R118A R118(L, I,V, M) R118K R118(N, Q) R118(D, E) R118(T, S) K139 K139A K139(I, L, V, M)K139R K139(N, Q) K139(D, E) K139(T, S) F180 F180A F180Y F180(I, L, V, M)In the examples provided infra, substituting an alanine would bepredicted to remove destabilizing residues for a naturally occurringbase at a mismatch. Alternatively, altering a charged polar residue witha pure hydrophobic residue may increase the stability or bindingaffinity of a naturally occurring base at a mismatch. In addition,changing a residue from a positive to negative charge may alter thespecificity of the enzyme, such that a natural base at a mismatch ispreferred. Since interactions between substrate and enzyme may involvecomplex hydrogen bond networks, altering two or more of the residueslisted in Table 7 may be required to obtain better activity of theenzyme to mismatches containing natural bases. Further, subtle changesin the catalytic residues, such as D43E or D105E, may assist incatalysis of naturally occurring base at a mismatch.

In analysis assuming dissimilar mismatch specificity among some membersof the EndoV family, 2 residues (G83 and I179) were identified thatcontain two separate sets of conserved sequence. For example, theresidues homologous to I179 of Thermotoga maritima Endo V, are either apolar-charged residue (K) or an aliaphatic residue (L, I, or V). If thisposition is involved in making an interaction with a DNA base at themismatch, the composition of the residue (polar and charged vs.hydrophobic) would influence which substrates are preferred. Theconserved set different from Thermotoga maritima endo V at the givenposition as the basis for initial mutagenesis (e.g., I179K) (Table 9)was used. TABLE 9 EndoV Mutants, Group 2. Add Remove DestabilizingStabilizing Residue Residue Modify Stability G83 G83(T, S) G83(N, Q)G83(D, E) G83(R, K) G83A G83(L, I, V, M) I179 I179A I179(R, K) I179(N,Q) I179(D, E) I179(T, S)

In addition to developing mutant Thermotoga maritima Endo V enzymes, thecloning of other thermostable Endo V enzymes provides a source ofalternative, but functionally similar, enzymes for use in the presentinvention. This invention is capable of utilizing other thermo stableEndoV enzymes, or other endonucleolytic enzymes as long as the productsare compatible with the thermo stable ligase step of the invention. Byutilizing a themostable Endo V with different substrate specificitiesfrom Thermotoga maritima Endo V, the capabilities of the presentinvention can be further extended.

Example 24 Mutation Scanning Using a Combined EndoV/DNA Ligase Assay

An analysis of p53 mutations in colon tumor DNA using a combination ofboth PCR/LDR/array and EndoV/Ligase mutation scanning proved superior toautomated sequencing (see Table 10). Of 23 samples shown to have 26 p53mutations by PCR/LDR combined with EndoV/ligase, 8 samples were missedby automated sequencing (65% true positive, 35% false negative).Mutation detection in five samples called positive with EndoV/Ligasemutation scanning, required gel purification of PCR fragments, andre-sequencing of both strands with manual reading. Significantly,EndoV/Ligase mutation scanning scored all four frameshift mutations,which accounts for almost 20% of the samples with p53 mutations. Suchframeshift mutations are beyond the detection capacity of commerciallyavailable p53 hybridization chips. TABLE 10 Detecting p53 mutations intumor samples: Comparisons of using a combined analysis with PCR/LDR andEndoV/ligase mutation scanning to Dideoxysequencing. Sample #: PCR/LDREndov/Ligase Dna sequencing, Resequencing of both (50 total) UniversalArray Mutation Scanning Automated Read strands, Manual read 53 (5) R175G2-A (5) R175 G2-A 55 (8) E285 G1-A (8) E285 G1-A (8) E285 G1-A 59 (8)R273 C-T (8) R273 C-T 60 (8) R282 C-T, (5) K164 A-T (5) K164 A-T, (8)R306 C-T (8) R306 C-T) 65 (5) R175 G2-A (5) R175 G2-A 66 (7) R248 G-A(7) R248 G-A (7) R248 G-A 67 (8) R282 C-T (8) R282 C-T (8) R282 C-T 68(6) Y205 A-T (6) Y205 A-T* 71 (8) R273 C-T (8) R273 C-T 73 (8) R282 C-T(5) Q167 delGT, (8) R282 C-T (5) Q167 delGT* (8) R282 C-T (8) R282 C-T77 (7) S261 T-G (7) S261 T-G 78 (7) R248 C-T (7) R248 C-T (7) R248 C-T79 (7) S240 delA (7) S240 delA* 80 (5) R175 G2-A (5) R175 G2-A 81 (5)R175 G2-A (5) R175 G2-A 84 (7) S261 T-G (7) S261 T-G 89 (6) Q192 C-T (6)Q192 C-T 90 (6) H214 delT (6) His 214 delT* 93 (5) R175 G2-A (5) R175G2-A 94 (6) H214 delT (6) His 214 delT* 96 (8) R273 G-A (8) R273 G-A (8)R273 G-A 97 (8) R276 C-G (8) R276 C-G 98 (8) R282 C-T (8) R282 C-T (8)R282 C-T (27 samples) No Mutation No Mutation Not determined# Notdetermined# Score 15/23 16/23 15/26 20/26 (16 known, 4 new, 4 (2resistant sites, deletions) observed 6 times) Percent 65% 70% 57% 77%Combined 100% 83% Adjusted 16/16 16/17 15/23 23/23 Percent 100% 94% 65%(100%)*Required gel purification of PCR product to obtain sequencing result.#Sequencing of 5 random samples of 27 called negative by PCR/LDR andEndoV/Ligase reveal no new mutations.

Mixing experiments using three different K-ras mutations demonstratedthat the Endo V/ligase reaction is also a sensitive assay: mutation canbe detected above background when 1 mutant sequence is diluted with 20wild-type sequences.

The colon tumor DNA was isolated from non-microdissected samples, withstromal cell infiltration from about 10%-50%. Mutation of one p53 alleleis invariably accompanied by loss of heterozygosity (“LOH”) of the otherallele, occurring either through chromosome loss or through mitoticnondisjunction (i.e. both mutant chromosomes migrate to the daughtercell). Thus, DNA from the above samples would have a range of mutantp53: wild-type allele of from about 90%:10% (for nondisjunction with 10%stroma) to 33%:67% (for chromosome loss with 50% stroma). When the abovesamples were reanalyzed for p53 mutations in pools of 3 samples, theEndoV mutation scanning assay could still identify the presence of allthe mutants. Several of the pooled sample bands were even stronger thanfrom individual samples (suggesting the original sample was mostlymutant DNA), while a few bands were weaker but could still be detected(suggesting the original sample had substantial stromal contamination).The ability to detect mutations in pools of 5 or even 10 samples wouldsignificantly improve cancer mutation scoring.

Example 25 Site-Directed Mutagenesis of Tma EndoV

Alanine scanning mutagenesis was performed at nine conserved positionsof Thermotoga maritima endoV to identify amino acid residues importantfor its endonuclease activity (Table 11). Alanine substitution at theDDE motif residues D43, E89, D110, abolish or substantially reduceinosine cleavage activity. These mutants also gain binding affinity todouble-stranded or single-stranded inosine substrate in the absence of ametal ion, suggesting that these residues are involved in thecoordination of catalytic Mg²⁺. Y80A, H116A, and, to a lesser extent,R88A, demonstrate reduced affinity to double-stranded or single-strandedinosine substrate or nicked product. The lack of tight binding to nickedinosine product accounts for an observed increase in turnover of inosinesubstrate since the product release is less rate-limiting. Y80A, R88A,K139A, and, to a lesser extent, H116A show reduced activity towards APand uracil substrates. Consistent with their location in or near thebase recognition pocket, these residues may play a role in substraterecognition. TABLE 11 DNA Cleavage Activity of Nine Tma EndovMutants^(a) Tma EndoV Mutants wt D43A Y80A R88A E89A D105A D110A H116AH125A K139A I/A ++ − ++ ++ + ++ − ++ ++ ++ A/U +++ − − − − +++ − + ++ −G/U +++ − − + − +++ − ++ +++ + T/U +++ − − ++ − +++ − ++ +++ + AP +++ −− − − +++ − + +++ − ssI ++ − ++ ++ + ++ − ++ ++ ++^(a)The cleavage reactions were performed at 65° C. for 30 minute in a20 μl reaction mixture containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2%glycerol, 5 mM MgCl₂, 10 nM DNA substrate, 100 nM purified Tma endoVmutants. The reactions were terminated by adding an equal volume ofGeneScan stop solution. The reaction mixtures were then heated at 94° C.for 2 min. and cooled on ice. Three microliters of samples were loadedonto a 10% GeneScan denaturing# polyacrylamide gel (Perkin Elmer). Electrophoresis was conducted at1500 voltage for 1 hr using an ABI 377 sequencer (Perkin Elmer). ss I:single stranded inosine-containing substrate. The number of plus signsrepresents the extent of cleavage of inosine-, uracil-, or APsite-containing strand.(+++) = about 10%;(++) = about 5%;(+) = about 2%;(−) no cleavage observed.

Site-specific mutagenesis was used to generate additional mutations atR88 (-→K, Q, E), H116 (-→Q, T, E), and K139 (-→Q, E). These mutantssubstantially lost activity using Mg²⁺ as cofactor. Four of the mutants(R88Q, R88E, H116Q, and H116T) cleaved certain substrates in Mn²⁺ betterthan wild-type enzyme does (See FIG. 20, lanes 4 & 6 compared to lane12, and Table 12). This result is not surprising since the mutantpresumably binds the substrate less well than wild-type enzyme, and,consequently, there is less non-specific binding and cleavage. The R88Qand R88E enzymes demonstrated a stronger preference for cleaving the Gbase in both G:A and A:G mismatched substrate than wild-type enzyme.Significantly, the preferred cleavage site of the “G” strand is nowpredominantly at the 3′ side of the mismatched base (See FIG. 20, Lane 1& 7, Table 12). Wild-type enzyme does not cleave the C strand in the A:Csubstrate, while the R88 mutant had medium level activity, with thepreferred cleavage site of the “C” strand now two bases from the 3′ endof the mismatch (Table 12). TABLE 12 Cleavage Intensities of varioussubstrates treated with wild-type and mutant Tma EndoV. Metal/ Substr.WT WT R88Q R88Q R88E R88E H116Q H116Q H116T H116T Mn2+ G: ++ A: +++ G:+++² A: ++⁴ G: ++++² A: ++⁴ G: +¹ A: ++++ G: A: +++⁴ A: w G: + A: ++⁴ G:+++ A: ++⁴ G: +++ A: ++³ G: A: ++⁴ G: A: ++++ G: +++ A: +++⁴ G: ++² A:++++ G: ++++ A: ++++ G: A: ++++ G: A: +++ G: ++ A: ++ G: ++³ A: +++ G:+++³ A: ++ G: A: +++ G: C: + A: +++ C: A: +++⁴ C: A: ++⁴ C: + A: ++ C: +A: +++ A: ++ C: A: ++⁴ C: ++⁶ A: ++⁴ C: +⁶ A: w C: w A: ++⁴ C: +⁶ T:++++ G: ++++ T: w G: +² T: +⁴ G: +++² T: +++ G: w T: ++ G: w G: +++ T:+++ G: +³ T: +⁴ G: ++³ T: +⁵ G: T: ++ G: T: +⁴ Mg2+ A: ++++ G: + A: G:A: w G: A: w G: A: ++ G: A: + G: w A: G: A: G: A: G: A: w G: Endo Vcleavage 0,1,2 nucleotides 3′ to the mismatch.

Superscripts (1-6) indicate cleavage patterns and relative intensities.No superscript indicates cleavage pattern was similar to wild-type (WT)enzyme. Overall cleavage intensities estimated based on uncut startingsubstrate. (++++) = about 30%; (+++) = about 20%; (++) = about 10%; (+)= about 5%; (w) = about 2%.

Both Tma EndoV H116Q and H116T exhibited almost exclusive activitytowards adenosine containing substrates (FIG. 20, Table 12, lanes 4 and6), with cleavage at the correct penultimate position. There was almostno activity towards the “G” containing strand, suggesting these mutantenzymes are the first step towards development of stronger enzymes forspecific mismatched sequences. The change in cleavage site for the R88and H116 mutants suggests significant plasticity in binding specificmismatched DNA by variant enzymes, further emphasizing the need formultiple crystal structures to understand non-classical cleavage forspecific mismatches.

Example 26 Alanine Scanning Mutagenesis

An overlapping extension PCR procedure was used for site-directedmutagenesis (Ho, S. et al., Gene 77:51-59 (1989), which is herebyincorporated by reference in its entirety). PCR products digested with apair of NdeI and SpeI were ligated to cloning vector pEV5 treated withthe same pair of restriction endonucleases. The ligated vectors weretransformed into E. coli strain AK53 (mrrB-, MM294). Plasmids containinginserts were reisolated and sequenced on a ABI sequencer usingDye-dideoxy terminator chemistry to identify mutated sequence and ensurethat the constructs were free of PCR error. Strains containing mutatedTma endonuclease V (nfi) genes were expressed in 5 ml of MOPS mediumsupplemented with 50 μg/ml ampicillin (Neidhart, F. C., et al., J.Bacteriol. 119(3):736-47 (1974), which is hereby incorporated byreference in its entirety) at 37° C. overnight. Cell pastes weresuspended in 300 μl of sonication buffer and sonicated 4×10 sec at 4° C.using a Sonifier Cell Disruptor 350 (Branson). After removing celldebris by centrifugation, the supernatants containing Tma endonuclease Vproteins were incubated at 70° C. for 15 min to inactivate hostproteins. The denatured proteins were separated from soluble Tmaendonuclease V proteins by centrifugation. The protein concentrations ofTma endonuclease V mutants were quantified by scanning a 12.5% SDS-PAGEgel loaded with known amounts of wild-type Tma endonuclease V. Partiallypurified proteins were diluted to 1 μM with 1× TaqI storage buffer andstored at −20° C. prior to use.

Example 27 DNA Cleavage Reaction for Assaying Variant Endonucleases

The fluorescence labeled deoxyoligonucleotide substrates were preparedas described (Huang, J., et al., Biochemistry 40(30):8738-8748 (2001),which is hereby incorporated by reference in its entirety). The sequenceof a typical inosine substrate (SEQ. ID. Nos. 1-2, respectively) is asfollows:                                              ↓ 5′-Fam-TA CCCCAG CGT CTG CGG TGT TGC GT   I AGT TGT CAT AGT TTG ATC CTC TAG TCT TGTTGC GGG TTC C-3′        3′-GGG GTC GCA GAC GCC ACA ACG CA   I TCA ACAGTA TCA AAC TAG GAG ATC AGA ACA ACG CCC-Tet-5′                                     ↑A nick event at the top strand generates a 27 nt labeled product whilethat at the bottom strand generates a 38 nt labeled product. Thecleavage reactions were performed at 65° C. for 30 minute in a 20 μlreaction mixture containing 10 mM HEPES (pH 7.4), 1 mM DTT, 2% glycerol,5 mM MgCl₂ unless otherwise specified, 10 nM DNA substrate, indicatedamount of purified Tma endonuclease V protein. The reaction wasterminated by adding an equal volume of GeneScan stop solution. Thereaction mixtures were then heated at 94° C. for 2 min and cooled onice. Three microliter of samples were loaded onto a 10% GeneScandenaturing polyacrylamide gel (Perkin Elmer). Electrophoresis wasconducted at 1500 voltage for 1 hr using an ABI 377 sequencer (PerkinElmer). Cleavage products and remaining substrates quantified using theGeneScan analysis software versions 2.1 or 3.0.

Example 28 Gel Mobility Shift Assay

The binding reaction mixture contains 100 nM double strandedfluorescence-labeled oligonucleotide DNA substrates, 5 mM MgCl₂ or CaCl₂or 2 mM EDTA, 20% glycerol, 10 mM HEPES (pH 7.4), 1 mM DTT, and 75 nM ofTma endonuclease V protein. The binding reactions were carried out at65° C. for 30 min. Samples was electrophoresed on a 6% nativepolyacrylamide gel in 1×TB buffer supplemented with 10 mM MgCl₂ or CaCl₂or 2 mM EDTA. The bound and free DNA species were analyzed usingFluorImager 595 (Molecular Dynamics) with the following settings: PMT at1000 Volt, excitation at 488 nm, emission at 530 nm (Filter 530 DF30).Data analysis was carried out using ImageQuaNT v4.1 (MolecularDynamics).

Example 29 DNA Cleavage by Variant Endonucleases

Tma endo V mutants were generated through an overlapping PCR procedureand partially purified through heat-treatment at 75° C. for 15 min. Theresulting mutant proteins are devoid of the indigenous E. coliendonuclease V (Huang, J., et al., Biochemistry 40(30):8738-8748 (2001),which is hereby incorporated by reference in its entirety). The cleavageassays were performed using oligonucleotide substrates fluorescentlylabeled at both strands. Previous studies have shown that the wild-typeTma endo V nicks at inosine sites. When the enzyme is in excess, itcleaves the complementary strand opposite of the inosine-containingstrand (Huang, J., et al., Biochemistry 40(30):8738-8748 (2001), whichis hereby incorporated by reference in its entirety). As expected forthe wild-type control, a blue band was observed which represented thetop inosine cleavage product and a green band which represented thebottom complementary strand cleavage product (FIG. 21A). Some lowermolecular weight nonspecific cleavage products were also observed. D43Aand D110A lost DNA endonuclease activity for cleaving either of thestrands. E89A exhibited limited cleavage of the inosine strand with nodetectable cleavage of the complementary strand. Y80A, R88A, H116A, andK139A maintained wt level cleavage activity towards inosine strand butlost complementary strand cleavage activity. D105A and H125A essentiallymaintained wt cleavage activity toward both the inosine-containingstrand and the complementary strand in the double-stranded substrate(FIG. 21A-C). Mn²⁺ enhanced non-specific cleavage activity but theoverall cleavage profiles of the mutants remained similar to Mg²⁺ (FIG.21B-C).

Example 30 Binding to Inosine Substrate by Variant Endonucleases

A fluorescence-based gel mobility shift assay was used to comparesubstrate binding among the nine alanine-substituted mutants (Huang, J.,et al., Biochemistry 40(30):8738-8748 (2001), which is herebyincorporated by reference in its entirety). As previously reported, thewt Tma endo V did not form a stable complex with the inosine substratein the absence of a metal cofactor ((Huang, J., et al., Biochemistry40(30):8738-8748 (2001), which is hereby incorporated by reference inits entirety) and FIG. 22A). Interestingly, the two catalyticallyinactive mutants D43A and D110A now formed a distinct complex with theinosine substrate. Although not observed in most mutational studiesperformed on endonucleases, the restriction endonuclease MunI does showsuch a peculiar property, i.e., elimination of the negative charges atsome metal binding residues confer binding specificity to the cognatesequence (Lagunavicius, A., et al., Biochemistry 36(37):11086-11092(1997), which is hereby incorporated by reference in its entirety). Itis suggested that the negative charges may cause electrostatic repulsionbetween the carboxylates and the phosphate in the absence of aneutralizing metal ion. By substituting the negatively charged Asp orGlu with Ala, the mutants become capable of binding to the cognatesequence even without a metal ion. A similar notion can be applied toexplain the binding behavior of D43A and D110A mutants, which impliesthat D43A and D110A are involved in the coordination of a divalent metalion required for catalysis.

In the presence of a metal cofactor such as Mg²⁺ or Ca²⁺, most ofmutants showed a binding affinity to the double-stranded inosinesubstrate comparable to that of the wt enzyme (FIG. 22B). Since Ca²⁺only supports binding but not catalysis (Huang, J., et al., Biochemistry40(30):8738-8748 (2001), which is hereby incorporated by reference inits entirety), the shifted bands represent an ES (enzyme-substrate)complex. The shifted bands observed with Mg²⁺, on the other hand, mayrepresent an enzyme-nicked product complex due to strand cleavage.Apparently, Y80A and H116A no longer bind to the intact inosinesubstrate at tightly as the wt enzyme as judged by gel shift data withCa²⁺ (FIG. 22B). Further, the gel shift data with Mg²⁺ suggested thatY80A and H116 may have reduced binding affinity to the nicked product aswell (FIG. 22C). To more definitively assess the impact ofalanine-substituted mutants had on endonuclease binding to the nickedproduct, gel mobility shift analysis was performed using a synthesizedinosine oligonucleotide substrate with a preexisting nick (FIG. 23). Itis evident from the gel shift experiments with Ca²⁺ that Y80A, R88A, andH116A failed to bind to the nicked inosine product as tightly as thewildtype (i.e. wt) enzyme (FIG. 23A). Using the catalytically competentmetal ion Mg²⁺ as the metal cofactor, H116A bound the nicked inosineproduct about half as tightly as the wt enzyme (FIG. 23B). Y80A did notmaintain bound to the nicked product with a long enough half-life toallow the detection of a distinct band shift (FIG. 23B-C). These resultssuggested that some endo V variants such as Y80A and H116A may haveaffected both ES and EP binding, while R88A may primarily have affectedproduct release.

A time course analysis was performed to assess how the altered bindingbehavior changed cleavage kinetics of the inosine-containing strand in adouble-stranded sequence. When the enzyme is in deficit (E:S=1:10), thewt enzyme only cleaved a limited amount of the substrate as it remainedbound to the nicked product (FIG. 24). Likewise, the mutant H125A, whichshowed wt level cleavage activity and binding affinity, also failed toturnover this inosine-containing double-stranded substrate. The threemutants Y80A, R88A, and H116A, which showed reduced affinities to thenicked inosine substrate by gel mobility shift analysis (FIG. 23), nowwere able to release the product and nicked the inosine strand in amultiple-turnover fashion (FIG. 24A). Most prominently, the cleavagereaction by Y80A proceeded close to completion with an apparent firstorder rate constant of 0.04 min⁻¹. The kinetic analysis, combined withthe binding data, suggests that some of the mutants (as represented byY80A) have changed the reaction kinetics by increasing the productrelease rate constant k₃ to facilitate multiple turnover (FIG. 24B).

Example 31 Cleavage and Binding of Single-Stranded Inosine Substrate byVariant Endonucleases

To assess the single-stranded endonuclease activity, cleavage wasexamined using fluorescently labeled (Tet) inosine-containingsingle-stranded oligonucleotide. Most of the endo V variants retained wtcleavage activity toward the single-stranded inosine-containingsubstrate (FIG. 25A). Consistent with the cleavage results obtainedusing the double-stranded inosine-containing substrate (FIG. 21), E89Ahad a significantly reduced cleavage activity, while D43A and D 110Awere inactive toward the single-stranded inosine-containing substrate(FIG. 25A). The binding of single-stranded inosine-containing substratealso required a metal cofactor for most of the mutants. D43A, D110A, andE89A, however, were able to bind to the single-strandedinosine-containing substrate without a metal ion (FIG. 25B), suggestinga similar interaction among the enzyme, inosine substrate, and metal ionfor both single-stranded or double-stranded substrates. The formation ofa distinct shifted band by E89A in the absence of a metal ion using thesingle-stranded inosine substrate suggests that E89 may play ananalogous role in metal binding similar to D43 and D110 (FIGS. 22A and25B).

The binding pattern of the single-stranded inosine-containing substratein the presence of Ca²⁺ is essentially identical to that of thedouble-stranded substrate, i.e., most of the mutants exhibited a bindingaffinity at least as strong as the wt enzyme. Y80A and H116A againshowed lower affinity to the inosine substrate (FIG. 25C). In thepresence of Mg²⁺, Y80A and H116A showed an intense lower molecularweight band with concurrent disappearance of the free DNA band,suggesting that most of the single-stranded substrate were cleaved (FIG.25D). Given the reduced binding seen with Ca²⁺, it is likely that thesetwo mutants no longer bind to the single-stranded nicked producttightly, thereby dissociating from the product. D43A, D110A, and E89Awere unable to cleave the inosine substrate or cleave at a much reducedrate (FIG. 25A). As expected, they formed a distinct stable complex asseen by mobility shift gel (FIG. 25D). However, they migrated fasterthan the wt and other catalytically active endo V mutants. One scenarioto explain the different migration patterns is that the binding of thewt enzyme and some other active mutants may cause bending of the nickedinosine product (FIG. 25D), resulting in a slower migration (Lane, D.,et al., Microbiol Rev 56(4):509-28 (1992), which is hereby incorporatedby reference in its entirety). D43A, D110A, and E89A mutants may havelost the ability to bend the DNA due to the loss of the negativecharges.

Example 32 Cleavage of Uracil and AP Site Substrates by VariantEndonucleases

Uracil endonuclease activity of Tma endo V mutants were assessed usingA/U, G/U, and T/U substrates. D43A, D110A, and E89A were inactive towardany of the uracil substrates, which is consistent with the proposedmetal binding role (FIG. 26A-C). D105A, which showed wild-type activitytowards various inosine substrates, maintained essentially wild-typeactivity with uracil-containing substrates. H125A cleaved the G/U andT/U substrates as well as the wild-type enzyme (FIG. 26B-C); however, ithad reduced activity against the perfectly base-paired substrate A/U(FIG. 26A). Y80A had minimal uracil cleavage activity. R88A, H116A, andK139A exhibited reduced uracil cleavage. The degree of uracil cleavageby these three mutants appeared to be affected by the nature of thebase-pair. When the substrate contained a Watson-Crick base-pair such asA/U (FIG. 26A), the uracil cleavage activity was lowest. When thesubstrate contained a mismatch such as G/U or T/U, the uracil cleavageactivity was enhanced (FIG. 26B-C). These results are in keeping withthe previous time course analysis of the wt enzyme using A/U and T/Usubstrates (Huang, J., et al., Biochemistry 40(30):8738-48 (2001), whichis hereby incorporated by reference in its entirety), suggesting that auracil in a locally distorted mismatch environment provides a bettersignal for base recognition.

AP endonuclease activity of Tma endo V mutants was assessed using theA/AP substrate. E. coli endonuclease IV is a thermostable 5′ APendonuclease (Ljungquist, S., J Biol Chem 252(9):p 2808-04 (1977), whichis hereby incorporated by reference in its entirety). To prevent theinterference of the host AP endonuclease, the wt and mutant endo V werefurther purified by HiTrap SP column chromatography (Amersham PharmaciaBiotech). Using column purified mutant proteins, AP endonucleaseactivity was only observed with D105A and H125A (FIG. 26D), both ofwhich had consistently showed wt DNA cleavage activities towards othersubstrates. Under the 65° C. assay conditions, the AP substrateunderwent spontaneous cleavage, generating a weak background as seen inthe control lane (FIG. 26D). Some marginal AP endonuclease activity maybe associated with H116A, but all other mutants abrogated AP cleavage.

Metal binding and active site (D43, D110, E89) One of the significantfindings from this study is identification of amino acid residuesinvolved in metal binding and catalysis. It is known from biochemicalstudies that endonuclease V is a metal-dependent DNA endonuclease(Huang, J., et al., Biochemistry 40(30):8738-48(2001) and Yao, M., etal., J. Biol Chem 269(23):16260-08 (1994), which are hereby incorporatedby reference in their entirety). The enzyme is most active with Mg²⁺ orMn²⁺ as the metal cofactor. Previous binding studies have demonstratedthat a divalent metal ion is not only required for catalysis but alsofor formation of a stable enzyme-substrate complex (Huang, J., et al.,Biochemistry 40(30):8738-48 (2001), which is hereby incorporated byreference in its entirety). For many endonucleases, a few negativelycharged amino acid residues, such as Asp and Glu, are involved in metalbinding (Cao, W., et al., J Biol Chem 273(49):33002-10 (1998); Kim, D.R., et al., Genes Dev 13(23):3070-80 (1999) and Koval, R. A., et al.,Curr Opin Chem Biol 3(5):578-83 (1999), which are hereby incorporated byreference in their entirety). Out of four such residues mutated, threeof them (D43, D110, E89) appear important for coordinating metal ion(s).D43A and D110A abolish the endonuclease activities towards allsubstrates tested, which include double-stranded and single-strandedinosine substrates (FIG. 21) and double-stranded uracil and AP sitesubstrates (FIG. 26). E89A had substantially reduced catalyticactivities. However, all three mutants retain wild-type binding affinitytowards inosine substrates, indicating their important role incatalysis. Most interestingly, D43A and D110A, and in some case E89A,bind to inosine substrates in the absence of a metal ion. D83A and E98Ain restriction endonuclease MunI have shown a similar binding behavior(Laugnavicius, A., et al., Biochemistry 36(37):11086-92 (1997), which ishereby incorporated by reference in its entirety). More recently, theMunI crystal structure complexed with cognate DNA positions D83A andE98A at the active site (Deibert, M., et al., Embo J 18(21):5805-16(1999), which is hereby incorporated by reference in its entirety). Itis likely that D43 and D110 in Tma endo V, and possibly E89, areinvolved in binding catalytic metal(s) for the hydrolysis of thescissile phosphate bond. Ca²⁺ competition has been used as a probe toassess the numbers of metal ions involved in DNA cleavage by EcoRV (Cao,W., et al., Biochim Biophys Acta 1546(1):253-60 (2001) and Vipond, I.B., et al., Biochemistry 34(2):697-704 (1995), which are herebyincorporated by reference in their entirety). A stimulatory effect byCa²⁺ on cognate site cleavage has been taken as an indication oftwo-metal catalytic mechanism. No enhancement of inosine cleavage wasobserved in Ca²⁺—Mg²⁺ competition. Thus, the number of metal ionsinvolved in strand cleavage in endonuclease V remains to be determined.

Y80 Alanine substitution at Y80 substantially reduced the affinity ofendo V to the double- and single-stranded inosine substrate (FIGS. 22and 25), as well as the nicked inosine product (FIG. 23). Evidently,weak binding helps endo V dissociate from the product, thereby allowingrapid turnover of the inosine substrate (FIG. 24). The loss of APendonuclease and uracil endonuclease activities may also be attributedto a weaker binding affinity (FIG. 26). These results suggest that Y80plays an important role in maintaining a stable complex between theenzyme and the repair-intermediate. Y80 may play such a role by makingdirect contact to the substrate and the product through base contact orphosphate backbone interaction, or by organizing a network of contactsmediated by other amino acid residues. The kinetic property ofaccelerated product release is analogous to R177A in human APendonuclease APE1 (Mol, C. D., et al., Nature 403(6768):451-56 (2000),which is hereby incorporated by reference in its entirety). In aDNA-bound structure, R177 makes a direct contact to the 3′ phosphate atthe AP site to lock APE1 onto the AP site (Mol, C. D., et al., Nature403(6768):451-56 (2000), which is hereby incorporated by reference inits entirety). Thus, a phosphate backbone contact may contribute totight product binding.

R88 and H116 R88A and H116A exhibit similar properties. Both of thesevariants have reduced affinity to the double-stranded inosine substrateand nicked product (FIGS. 22-23), which results in a higher turnover(FIG. 24). They maintain wt level single-stranded inosine cleavageactivity (FIG. 25). The major difference between R88A and H116A is aturacil substrates. H116A is about 9-fold more active than R88A for G/Usubstrate and 5-fold more active for T/U substrate (FIG. 26).Apparently, R88 plays a more important role in uracil recognition.

K139 Alanine substitution at K139 has little effect on binding (FIGS.22-23, 25). K139A maintains wt level cleavage activity toward thedouble-stranded inosine substrate I/A (FIG. 21). Similar to the wtenzyme, K139A remains bound even after cleaving inosine substrate due totight product binding (FIG. 24). The main effects of K139A substitutionon endo V is at cleavage of non-inosine substrates. In addition to lackof AP endonuclease activity, K139A has a substantially reduced uracilendonuclease activity (FIG. 26). A more detailed kinetic analysis isrequired to understand the precise catalytic role K139 may play withdifferent substrates.

As the first step to elucidate the structural basis of endonuclease Vcatalytic and recognition mechanism, an alanine scanning mutagenesis wascarried out at nine conserved positions of Tma endonuclease V. D43 andD110, along with E89 may coordinate metal ion(s) for the scissilephosphate bond hydrolysis and are part of the active site. Y80 makessubstantial contribution to substrate and nicked product binding. Whilethe lack of strong binding to substrate may account for loss of AP siteand uracil cleavage activity, this same property also changes thekinetics of cleaving inosine-containing substrates, such that theproduct release step is no longer rate limiting. R88A and H116A affectbinding steps as well, resulting in diminishing endonuclease activitiestoward non-inosine substrates. The tight binding by K139A limits theturnover of inosine substrates. The lack of AP site and uracil cleavagesuggests that K139A may affect steps other than substrate or productbinding. A full understanding of the mutational effects described hereawaits determination of the endo V-DNA complex structure.

Generation of additional Tma endonuclease V mutants at the abovementioned positions revealed four of these variants (R88Q, R88E, H116Q,and H116T) which preferentially nicked or cleaved at least oneheteroduplexed DNA containing mismatched bases better than wild-typeenzyme in selected buffer conditions. The R88Q and R88E enzymesdemonstrated a stronger preference for cleaving the G base in both G:Aand A:G mismatched substrate than wild-type endonuclease. The H116Q andH116T enzymes exhibited almost exclusive activity towards “A” basecontaining substrates. There was almost no activity towards the “G”containing strand, suggesting these variant enzymes are proof ofprinciple that stronger enzymes for specific mismatched sequences can bedeveloped. Such enzymes will improve signal-to-noise in scoring ofunknown mutations and polymorphisms, both in pooling experiments, andfor mutations in difficult sequence environments.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions and the like can bemade without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method for identifying a mutant nucleic acid sequence differing byone or more single-base changes, insertions, or deletions, from a normaltarget nucleotide sequences, said method comprising: providing a samplepotentially containing the normal target nucleotide sequence as well asthe mutant nucleic acid sequence; providing two labeled oligonucleotideprimers suitable for hybridization on complementary strands of thetarget nucleotide sequence and the mutant nucleic acid sequence;providing a polymerase; blending the sample, the labeled oligonucleotideprimers, and the polymerase to form a polymerase chain reaction mixture;subjecting the polymerase chain reaction mixture to one or morepolymerase chain reaction cycles comprising a hybridization treatment,wherein oligonucleotide primers can hybridize to the target nucleotidesequence and/or the mutant nucleic acid sequence, an extensiontreatment, wherein the hybridized oligonucleotide primer is extended toform an extension product complementary to the target nucleotidesequence and/or the mutant nucleic acid sequence to which theoligonucleotide primer is hybridized, and a denaturation treatment,wherein hybridized nucleic acid sequences are separated; inactivatingthe polymerase; denaturing the polymerase chain reaction extensionproducts; annealing the polymerase chain reaction extension products toform heteroduplexed products potentially containing the normal targetnucleotide sequence and the mutant nucleic acid sequence; providing anendonuclease which preferentially nicks or cleaves heteroduplexed DNA ata location one base away from mismatched base pairs; blending theheteroduplexed products and the endonuclease to form an endonucleasecleavage reaction mixture; incubating the endonuclease cleavage reactionmixture so that the endonuclease preferentially nicks or cleavesheteroduplexed products at a location one base away from mismatched basepairs; providing a ligase; blending the potentially nicked or cleavedheteroduplexed products and the ligase to form a ligase resealingreaction mixture; incubating the ligase resealing reaction mixture toseal the nicked heteroduplexed products at perfectly matched base pairsbut with substantially no resealing of nicked heteroduplexed products atlocations adjacent to mismatched base pairs; separating productsresulting from said incubating the ligase resealing reaction mixture bysize or electrophoretic mobility; and detecting the presence of thenormal target nucleotide sequence and the mutant nucleic acid sequencein the sample by distinguishing the separated products resulting fromsaid incubating the ligase resealing reaction mixture, wherein theendonuclease is a mutant endonuclease which preferentially nicks orcleaves at least one heteroduplexed DNA, containing mismatched bases,better than a wild-type endonuclease.
 2. A method according to claim 1,wherein one of the mismatched bases in at least one of theheteroduplexed DNA is an “A”.
 3. A method according to claim 1, whereinone of the mismatched bases in at least one of the heteroduplexed DNA isa “G”.
 4. A method according to claim 1, wherein the endonuclease is amutant endonuclease V from Thermotoga maritima containing either: (1) aY80A residue change; (2) a Y80F residue change; (3) either a Y80L, Y80I,Y80V or Y80M residue change; (4) an R88A residue change; (5) an R88L,R88I, R88V, or R88M residue change; (6) an R88K residue change; (7) anR88N or R88Q residue change; (8) an R88D or R88E residue change; (9) anR88T or R88S residue change; (10) a E89A residue change; (11) a E89L,E89I, E89V, or E89M residue change; (12) a E89D residue change; (13) aE89N or E89Q residue change; (14) a E89R or E89K residue change; (15) aE89T or E89S residue change; (16) a H116A residue change; (17) a H116L,H116I, H116V, or H116M residue change; (18) a H116K or H116R residuechange; (19) a H116N or H116Q residue change; (20) a H116T or H116Sresidue change; (21) a K139A residue change; (22) a K139L, K139I, K139V,or K139M residue change; (23) a K139R residue change; (24) a K139N orK139Q residue change; (25) a K139D or K139E residue change; (26) a K139Tor K139S residue change; (27) a D43A residue change; (28) a D43E residuechange; (29) a D105A residue change; (30) a D105E residue change; (31)an F46A residue change; (32) an F46Y residue change; (33) an F46L, F46I,F46V, or F46M residue change; (34) an R118A residue change; (35) anR118L, R118I, R118V, or R118M residue change; (36) an R118K residuechange; (37) an R118N or R118Q residue change; (38) an R118D or R118Eresidue change; (39) an R118T or R118S residue change; (40) a F180Aresidue change; (41) a F180Y residue change; (42) a F180L, F180I, F180V,or F180M residue change; (43) a G83A residue change; (44) a G83L, G83I,G83V, or G83M residue change; (45) a G83K or G83R residue change; (46) aG83N or G83Q residue change; (47) a G83D or G83E residue change; (48) aG83T or G83S residue change; (49) an I179A residue change; (50) an I179Kor I179R residue change; (51) an I179N or I179Q residue change; (52) anI179D or I179E residue change; (53) an I179T or I179S residue change;(54) a D110A residue change; or (55) an H125A residue change.
 5. Amethod for identifying a mutant nucleic sequence differing by one ormore single-base changes, insertions, or deletions from a normal targetnucleic acid sequence, said method comprising: providing a samplepotentially containing the mutant nucleic acid sequence but notnecessarily the normal target nucleic acid sequence; providing astandard containing the normal target nucleic acid sequence; providingtwo labeled oligonucleotide primers suitable for hybridization oncomplementary strands of the mutant nucleic acid sequence; providing apolymerase; blending the sample, the standard, the labeledoligonucleotide primers, and the polymerase to form a first polymerasechain reaction mixture; subjecting the first polymerase chain reactionmixture to one or more polymerase chain reaction cycles comprising ahybridization treatment, wherein the labeled oligonucleotide primers canhybridize to the mutant nucleic acid sequence, an extension treatment,wherein the hybridized oligonucleotide primer is extended to form anextension product complementary to the mutant nucleic acid sequence towhich the oligonucleotide primer is hybridized, and a denaturationtreatment, wherein hybridized nucleic acid sequences are separated;inactivating the polymerase; providing the normal target nucleic acidsequence; blending the normal target nucleic acid sequence, the labeledoligonucleotide primers, and the polymerase to form a second polymerasechain reaction mixture; subjecting the second polymerase chain reactionmixture to one or more polymerase chain reaction cycles comprising ahybridization treatment, wherein the labeled oligonucleotide primers canhybridize to the normal target nucleic acid sequence, an extensiontreatment, wherein the hybridized oligonucleotide primer is extended toform an extension product complementary to the normal target nucleicacid sequence to which the oligonucleotide primer is hybridized, and adenaturation treatment, wherein hybridized nucleic acid sequences areseparated; inactivating the polymerase; blending the first and secondpolymerase chain reaction extension products; denaturing the first andsecond polymerase chain reaction extension products; annealing the firstand second polymerase chain reaction extension products to formheteroduplexed products potentially containing the normal target nucleicacid sequence and the mutant nucleic acid sequence; providing anendonuclease which preferentially nicks or cleaves heteroduplexed DNA ata location one base away from mismatched base pairs; blending theheteroduplexed products and the endonuclease to form an endonucleasecleavage reaction mixture; incubating the endonuclease cleavage reactionmixture so that the endonuclease preferentially nicks or cleavesheteroduplexed products at a location one base away from mismatched basepairs; providing a ligase; blending the potentially nicked or cleavedheteroduplexed products and the ligase to form a ligase resealingreaction mixture; incubating the ligase resealing reaction mixture toseal the nicked heteroduplexed products at perfectly matched base pairsbut with substantially no resealing of nicked heteroduplexed products atlocations adjacent to mismatched base pairs; separating productsresulting from said incubating the ligase resealing reaction mixture bysize or electrophoretic mobility; and detecting the presence of thenormal target nucleic acid sequence and the mutant nucleic acid sequencetarget nucleotide in the sample by distinguishing the separated productsresulting from said incubating the ligase resealing reaction mixture,wherein the endonuclease is a mutant endonuclease which preferentiallynicks or cleaves at least one heteroduplexed DNA, containing mismatchedbases, better than a wild-type endonuclease.
 6. A method according toclaim 5, wherein one of the mismatched bases in at least one of theheteroduplexed DNA is an “A”.
 7. A method according to claim 5, whereinone of the mismatched bases in at least one of the heteroduplexed DNA isa “G”.
 8. A method according to claim 5, wherein the endonuclease is amutant endonuclease V from Thermotoga maritima containing either: (1) aY80A residue change; (2) a Y80F residue change; (3) either a Y80L, Y80I,Y80V or Y80M residue change; (4) an R88A residue change; (5) an R88L,R88I, R88V, or R88M residue change; (6) an R88K residue change; (7) anR88N or R88Q residue change; (8) an R88D or R88E residue change; (9) anR88T or R88S residue change; (10) a E89A residue change; (11) a E89L,E89I, E89V, or E89M residue change; (12) a E89D residue change; (13) aE89N or E89Q residue change; (14) a E89R or E89K residue change; (15) aE89T or E89S residue change; (16) a H116A residue change; (17) a H116L,H116I, H116V, or H116M residue change; (18) a H116K or H116R residuechange; (19) a H116N or H116Q residue change; (20) a H116T or H116Sresidue change; (21) a K139A residue change; (22) a K139L, K139I, K139V,or K139M residue change; (23) a K139R residue change; (24) a K139N orK139Q residue change; (25) a K139D or K139E residue change; (26) a K139Tor K139S residue change; (27) a D43A residue change; (28) a D43E residuechange; (29) a D105A residue change; (30) a D105E residue change; (31)an F46A residue change; (32) an F46Y residue change; (33) an F46L, F46I,F46V, or F46M residue change; (34) an R118A residue change; (35) anR118L, R118I, R118V, or R118M residue change; (36) an R118K residuechange; (37) an R118N or R118Q residue change; (38) an R118D or R118Eresidue change; (39) an R118T or R118S residue change; (40) a F180Aresidue change; (41) a F180Y residue change; (42) a F180L, F180I, F180V,or F180M residue change; (43) a G83A residue change; (44) a G83L, G83I,G83V, or G83M residue change; (45) a G83K or G83R residue change; (46) aG83N or G83Q residue change; (47) a G83D or G83E residue change; (48) aG83T or G83S residue change; (49) an I179A residue change; (50) an I179Kor I179R residue change; (51) an I179N or I179Q residue change; (52) anI179D or I179E residue change; (53) an I179T or I179S residue change;(54) a D110A residue change; or (55) an H125A residue change.
 9. Amutant endonuclease V from Thermotoga maritima containing either: (1) aY80A residue change; (2) a Y80F residue change; (3) either a Y80L, Y80I,Y80V or Y80M residue change; (4) an R88A residue change; (5) an R88L,R88I, R88V, or R88M residue change; (6) an R88K residue change; (7) anR88N or R88Q residue change; (8) an R88D or R88E residue change; (9) anR88T or R88S residue change; (10) a E89A residue change; (11) a E89L,E89I, E89V, or E89M residue change; (12) a E89D residue change; (13) aE89N or E89Q residue change; (14) a E89R or E89K residue change; (15) aE89T or E89S residue change; (16) a H116A residue change; (17) a H116L,H116I, H116V, or H116M residue change; (18) a H116K or H116R residuechange; (19) a H116N or H116Q residue change; (20) a H116T or H116Sresidue change; (21) a K139A residue change; (22) a K139L, K139I, K139V,or K139M residue change; (23) a K139R residue change; (24) a K139N orK139Q residue change; (25) a K139D or K139E residue change; (26) a K139Tor K139S residue change; (27) a D43A residue change; (28) a D43E residuechange; (29) a D105A residue change; (30) a D105E residue change; (31)an F46A residue change; (32) an F46Y residue change; (33) an F46L, F46I,F46V, or F46M residue change; (34) an R118A residue change; (35) anR118L, R118I, R118V, or R118M residue change; (36) an R118K residuechange; (37) an R118N or R118Q residue change; (38) an R118D or R118Eresidue change; (39) an R118T or R118S residue change; (40) a F180Aresidue change; (41) a F180Y residue change; (42) a F180L, F180I, F180V,or F180M residue change; (43) a G83A residue change; (44) a G83L, G83I,G83V, or G83M residue change; (45) a G83K or G83R residue change; (46) aG83N or G83Q residue change; (47) a G83D or G83E residue change; (48) aG83T or G83S residue change; (49) an I179A residue change; (50) an I179Kor I179R residue change; (51) an I179N or I179Q residue change; (52) anI179D or I179E residue change; (53) an I179T or I179S residue change;(54) a D110A residue change; or (55) an H125A residue change.
 10. Amutant endonuclease V according to claim 9, wherein the endonuclease Vcontains a Y80A residue change.
 11. A mutant endonuclease V according toclaim 9, wherein the endonuclease V contains a Y80F residue change. 12.A mutant endonuclease V according to claim 9, wherein the endonuclease Vcontains a Y80L, Y80I, Y80V or Y80M residue change.
 13. A mutantendonuclease V according to claim 9, wherein the endonuclease V containsan R88A residue change.
 14. A mutant endonuclease V according to claim9, wherein the endonuclease V contains an R88L, R88I, R88V, or R88Mresidue change.
 15. A mutant endonuclease V according to claim 9,wherein the endonuclease V contains an R88K residue change.
 16. A mutantendonuclease V according to claim 9, wherein the endonuclease V containsan R88N or R88Q residue change.
 17. A mutant endonuclease V according toclaim 9, wherein the endonuclease V contains an R88D or R88E residuechange.
 18. A mutant endonuclease V according to claim 9 wherein theendonuclease V contains an R88T or R88S residue change.
 19. A mutantendonuclease V according to claim 9, wherein the endonuclease V containsan E89A residue change.
 20. A mutant endonuclease V according to claim9, wherein the endonuclease V contains an E89L, E89I, E89V, or E89Mresidue change.
 21. A mutant endonuclease V according to claim 9,wherein the endonuclease V contains an E89D residue change.
 22. A mutantendonuclease V according to claim 9, wherein the endonuclease V containsan E89N or E89Q residue change.
 23. A mutant endonuclease V according toclaim 9, wherein the endonuclease V contains an E89R or E89K residuechange.
 24. A mutant endonuclease V according to claim 9, wherein theendonuclease V contains an E89T or E89S residue change.